Production and purification of IL-29

ABSTRACT

The expression vectors and methods using an  E. coli  expression system for the large scale production of IL-29 are described. The vectors utilize the IL-29 coding sequence with specific changes in nucleotides in order to optimize codons and mRNA secondary structure for translation in  E. coli . Also included are methods of producing, purifying and pegylating an IL-29 polypeptide.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is divisional of U.S. patent application Ser.No. 12/537,935, filed Aug. 7, 2009, now U.S. Pat. No. 7,759,092, whichis a continuation of U.S. patent application Ser. No. 11/538,688, filedOct. 4, 2006, now abandoned, which claims the benefit of U.S. PatentApplication Ser. No. 60/723,544, filed Oct. 4, 2005, all of which areherein incorporated by reference in their entireties.

BACKGROUND OF THE INVENTION

The increased availability and identification of genes from human andother genomes has led to an increased need for efficient expression andpurification of recombinant proteins. The expression of proteins inbacteria is by far the most widely used approach for the production ofcloned genes. For many reasons, expression in bacteria is preferred toexpression in eukaryotic cells. For example, bacteria are much easier togrow than eukaryotic cells. More specifically, the availability of awealth of sophisticated molecular genetic tools and thousands of mutantsmake E. coli, as an expression host, extremely useful for proteinproduction. However, the high-level production of functional proteins inE. coli, especially those from eukaryotic sources has often beendifficult.

IL-28A, IL-28B, and IL-29 comprise a recently discovered new family ofproteins that have sequence homology to type I interferons and genomichomology to IL-10. This new family is fully described in co-owned PCTapplication WO 02/086087 and Sheppard et al., Nature Immunol 4:63-68,2003. Functionally, IL-28A, IL-28B and IL-29 resemble type I INFs intheir ability to induce an antiviral state in cells but, unlike type IIFNs, they do not display antiproliferative activity against certain Bcell lines.

Recombinant IL-29 has been produced in prokaryotic cells, in particularE. coli. The resulting bacterial produced protein is not glycosylated,and is produced in an aggregated state. Production of IL-29 from E. colirequires that the aggregated proteins be solubilized from the insolubleinclusion bodies and renatured or refolded. Without renaturation, thespecific activity of the recombinant protein will be significantlyreduced.

Despite advances in the expression of recombinant proteins in bacterialhosts, there exists a need for improved methods for producingbiologically active and purified recombinant IL-29 proteins inprokaryotic systems which result in higher yields for proteinproduction. These and other aspects of the invention will become evidentupon reference to the following detailed description.

DESCRIPTION OF THE INVENTION Definitions

In the description that follows, a number of terms are used extensively.The following definitions are provided to facilitate understanding ofthe invention.

Unless otherwise specified, “a,” “an,” “the,” and “at least one” areused interchangeably and mean one or more than one.

As used herein, “nucleic acid” or “nucleic acid molecule” refers topolynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid(RNA), oligonucleotides, fragments generated by the polymerase chainreaction (PCR), and fragments generated by any of ligation, scission,endonuclease action, and exonuclease action. Nucleic acid molecules canbe composed of monomers that are naturally-occurring nucleotides (suchas DNA and RNA), or analogs of naturally-occurring nucleotides (e.g.,α-enantiomeric forms of naturally-occurring nucleotides), or acombination of both. Modified nucleotides can have alterations in sugarmoieties and/or in pyrimidine or purine base moieties. Sugarmodifications include, for example, replacement of one or more hydroxylgroups with halogens, alkyl groups, amines, and azido groups, or sugarscan be functionalized as ethers or esters. Moreover, the entire sugarmoiety can be replaced with sterically and electronically similarstructures, such as aza-sugars and carbocyclic sugar analogs. Examplesof modifications in a base moiety include alkylated purines andpyrimidines, acylated purines or pyrimidines, or other well-knownheterocyclic substitutes. Nucleic acid monomers can be linked byphosphodiester bonds or analogs of such linkages. Analogs ofphosphodiester linkages include phosphorothioate, phosphorodithioate,phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate,phosphoranilidate, phosphoramidate, and the like. The term “nucleic acidmolecule” also includes so-called “peptide nucleic acids,” whichcomprise naturally-occurring or modified nucleic acid bases attached toa polyamide backbone. Nucleic acids can be either single stranded ordouble stranded.

The term “complement of a nucleic acid molecule” refers to a nucleicacid molecule having a complementary nucleotide sequence and reverseorientation as compared to a reference nucleotide sequence.

An “enhancer” is a type of regulatory element that can increase theefficiency of transcription, regardless of the distance or orientationof the enhancer relative to the start site of transcription.

“Heterologous DNA” refers to a DNA molecule, or a population of DNAmolecules, that does not exist naturally within a given host cell. DNAmolecules heterologous to a particular host cell may contain DNA derivedfrom the host cell species (i.e., endogenous DNA) so long as that hostDNA is combined with non-host DNA (i.e., exogenous DNA). For example, aDNA molecule containing a non-host DNA segment encoding a polypeptideoperably linked to a host DNA segment comprising a transcriptionpromoter is considered to be a heterologous DNA molecule. Conversely, aheterologous DNA molecule can comprise an endogenous gene operablylinked with an exogenous promoter. As another illustration, a DNAmolecule comprising a gene derived from a wild-type cell is consideredto be heterologous DNA if that DNA molecule is introduced into a mutantcell that lacks the wild-type gene.

The term “contig” denotes a nucleic acid molecule that has a contiguousstretch of identical or complementary sequence to another nucleic acidmolecule. Contiguous sequences are said to “overlap” a given stretch ofa nucleic acid molecule either in their entirety or along a partialstretch of the nucleic acid molecule.

“Complementary DNA (cDNA)” is a single-stranded DNA molecule that isformed from an mRNA template by the enzyme reverse transcriptase.Typically, a primer complementary to portions of mRNA is employed forthe initiation of reverse transcription. Those skilled in the art alsouse the term “cDNA” to refer to a double-stranded DNA moleculeconsisting of such a single-stranded DNA molecule and its complementaryDNA strand. The term “cDNA” also refers to a clone of a cDNA moleculesynthesized from an RNA template.

An “isolated nucleic acid molecule” is a nucleic acid molecule that isnot integrated in the genomic DNA of an organism. For example, a DNAmolecule that encodes a growth factor that has been separated from thegenomic DNA of a cell is an isolated DNA molecule. Another example of anisolated nucleic acid molecule is a chemically-synthesized nucleic acidmolecule that is not integrated in the genome of an organism. A nucleicacid molecule that has been isolated from a particular species issmaller than the complete DNA molecule of a chromosome from thatspecies.

“Linear DNA” denotes non-circular DNA molecules with free 5′ and 3′ends. Linear DNA can be prepared from closed circular DNA molecules,such as plasmids, by enzymatic digestion or physical disruption.

A “promoter” is a nucleotide sequence that directs the transcription ofa structural gene. Typically, a promoter is located in the 5′ non-codingregion of a gene, proximal to the transcriptional start site of astructural gene. Sequence elements within promoters that function in theinitiation of transcription are often characterized by consensusnucleotide sequences. These promoters include “inducible promoters”, forexample, but are not limited to, IPTG-inducible promoters (such as thetac promoters; trc promoters; lac promoters; bacteriophage T7, T3, T5promoters; and nprM-lac promoters), trp promoters, phoA promoters, recApromoters, cspA promoters, tetA promoters, and bacteriophage λp_(L). SeeSambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001. Additionof an “inducing agent”, e.g., isopropyl thiogalactopyranoside (IPTG) foran IPTG-inducible promoter, will induce expression of the gene or genesunder the control of the IPTG-inducible promoter. A typical promoterwill have three components, consisting of consensus sequences at −35 and−10 with a sequence of between 16 and 19 nucleotides between them(Lisset, S. and Margalit, H., Nucleic Acids Res. 21: 1512, 1993).Promoters of this sort include the lac, trp, trp-lac (tac) and trp-lac(trc) promoters. If a promoter is an inducible promoter, then the rateof transcription increases in response to an inducing agent. Incontrast, the rate of transcription is not regulated by an inducingagent if the promoter is a constitutive promoter. Repressible promotersare also known.

A “core promoter” contains essential nucleotide sequences for promoterfunction, including the start of transcription. By this definition, acore promoter may or may not have detectable activity in the absence ofspecific sequences that may enhance the activity or confer tissuespecific activity.

A “regulatory element” is a nucleotide sequence that modulates theactivity of a core promoter. For example, a eukaryotic regulatoryelement may contain a nucleotide sequence that binds with cellularfactors enabling transcription exclusively or preferentially inparticular cells, tissues, or organelles. These types of regulatoryelements are normally associated with genes that are expressed in a“cell-specific,” “tissue-specific,” or “organelle-specific” mannerBacterial promoters have regulatory elements that bind and modulate theactivity of the core promoter, such as operator sequences that bindactivator or repressor molecules.

A “cloning vector” is a nucleic acid molecule, such as a plasmid,cosmid, or bacteriophage, which has the capability of replicatingautonomously in a host cell. Cloning vectors typically contain one or asmall number of restriction endonuclease recognition sites that allowinsertion of a nucleic acid molecule in a determinable fashion withoutloss of an essential biological function of the vector, as well asnucleotide sequences encoding a marker gene that is suitable for use inthe identification and selection of cells transformed with the cloningvector. Marker genes typically include genes that provide resistance toantibiotic.

An “expression vector” is a nucleic acid molecule encoding a gene thatis expressed in a host cell. Typically, an expression vector comprises atranscriptional promoter, a gene, an origin of replication, a selectablemarker, and a transcriptional terminator. Gene expression is usuallyplaced under the control of a promoter, and such a gene is said to be“operably linked to” the promoter. Similarly, a regulatory element and acore promoter are operably linked if the regulatory element modulatesthe activity of the core promoter. An expression vector may also beknown as an expression construct.

A “recombinant host” is a cell that contains a heterologous nucleic acidmolecule, such as a cloning vector or expression vector.

The term “expression” refers to the biosynthesis of a gene product. Forexample, in the case of a structural gene, expression involvestranscription of the structural gene into mRNA and the translation ofmRNA into one or more polypeptides.

The term “secretory signal sequence” denotes a DNA sequence that encodesa peptide (a “secretory peptide”) that, as a component of a largerpolypeptide, directs the larger polypeptide through a secretory pathwayof a cell in which it is synthesized. The larger polypeptide is commonlycleaved to remove the secretory peptide during transit through thesecretory pathway.

A “polypeptide” is a polymer of amino acid residues joined by peptidebonds, whether produced naturally or synthetically. Polypeptides of lessthan about 10 amino acid residues are commonly referred to as“peptides.”

A “protein” is a macromolecule comprising one or more polypeptidechains. A protein may also comprise non-peptidic components, such ascarbohydrate groups. Carbohydrates and other non-peptidic substituentsmay be added to a protein. Proteins are defined herein in terms of theiramino acid backbone structures; substituents such as carbohydrate groupsand non-peptidic groups are generally not specified, but may be presentnonetheless.

A peptide or polypeptide encoded by a non-host DNA molecule is a“heterologous” peptide or polypeptide.

An “isolated polypeptide” is a polypeptide that is essentially free fromcontaminating cellular components, such as carbohydrate, lipid, or otherproteinaceous impurities associated with the polypeptide in nature.Typically, a preparation of isolated polypeptide contains thepolypeptide in a highly purified form, i.e., at least about 80% pure, atleast about 90% pure, at least about 95% pure, greater than 95% pure, orgreater than 99% pure. One way to show that a particular proteinpreparation contains an isolated polypeptide is by the appearance of asingle band following sodium dodecyl sulfate (SDS)-polyacrylamide gelelectrophoresis of the protein preparation and Coomassie Brilliant Bluestaining of the gel. However, the term “isolated” does not exclude thepresence of the same polypeptide in alternative physical forms, such asdimers or alternatively glycosylated or derivatized forms.

The terms “amino-terminal” or “N-terminal” and “carboxyl-terminal” or“C-terminal” are used herein to denote positions within polypeptides.Where the context allows, these terms are used with reference to aparticular sequence or portion of a polypeptide to denote proximity orrelative position. For example, a certain sequence positionedcarboxyl-terminal to a reference sequence within a polypeptide islocated proximal to the carboxyl terminus of the reference sequence, butis not necessarily at the carboxyl terminus of the complete polypeptide.

A “fusion protein” is a hybrid protein expressed by a nucleic acidmolecule comprising nucleotide sequences of at least two genes.

The term “affinity tag” is used herein to denote a polypeptide segmentthat can be attached to a second polypeptide to provide for purificationor detection of the second polypeptide or provide sites for attachmentof the second polypeptide to a substrate. In principal, any peptide orprotein for which an antibody or other specific binding agent isavailable can be used as an affinity tag. Affinity tags include apoly-histidine tract, protein A (Nilsson et al., EMBO J. 4:1075 (1985);Nilsson et al., Methods Enzymol. 198:3 (1991)), glutathione Stransferase (Smith and Johnson, Gene 67:31 (1988)), Glu-Glu affinity tag(Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952 (1985)),substance P, FLAG peptide (Hopp et al., Biotechnology 6:1204 (1988)),streptavidin binding peptide, or other antigenic epitope or bindingdomain. See, in general, Ford et al., Protein Expression andPurification 2:95 (1991). DNA molecules encoding affinity tags areavailable from commercial suppliers (e.g., Pharmacia Biotech,Piscataway, N.J.).

The term “isotonic” is used herein for its conventional meaning, whichis a tonicity equal to that of blood, equivalent to a 0.9% solution ofNaCl. “An isotonic amount” of a salt is that amount required to make asolution isotonic or to produce an isotonic solution upon reconstitutionof a lyophilized preparation.

Concentrations are specified herein in units of molarity or % w/v ofliquid compositions. When the composition is in the form of alyophilized powder, the concentrations of the respective components willbe such as to provide the specified concentration on reconstitution ofthe powder.

Due to the imprecision of standard analytical methods, molecular weightsand lengths of polymers are understood to be approximate values. Whensuch a value is expressed as “about” X or “approximately” X, the statedvalue of X will be understood to be accurate to ±10%.

The present invention provides an expression vector for producing anIL-29 polypeptide comprising the operably linked elements of aprokaryotic origin of replication, a transcriptional initiation DNAelement, and polynucleotide sequence selected from the group consistingof SEQ ID NOs:1, 3, 5, 7, 9 and 11 and a transcriptional terminator. Inanother aspect, the expression vector is the vector pTAP440 or pTAP395.Optionally, the expression vector may further include a selectablemarker, such as kanamycin.

In another aspect, the present invention provides prokaryotic host cellstransformed with expression vectors comprising a polynucleotide sequenceencoding an IL-29 polypeptide (e.g., SEQ ID NOs:2, 4, 6, 8, 10, 12, andbiologically active mutants), vector pTAP440 or vector pTAP395. In otherembodiments, the host strain is E. coli strain W3110, zGOLD1, or zGOLD5.

In another aspect, the present invention provides methods for producingan IL-29 polypeptide under conditions wherein the IL-29 polypeptide isexpressed. In one embodiment, the method comprises culturing a host cellexpressing an IL-29 polypeptide after being transformed with pTAP440 orpTAP395. In another embodiment, the method comprises culturing a hostcell transformed with an expression vector comprising a polypeptideselected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, andbiologically active mutants. The method also comprises recovering thehost cells from the growth medium, and then isolating the IL-29polypeptide from the host cells.

In other aspects, the present invention provides a method of producingan IL-29 polypeptide comprising the steps as described above, in a fedbatch fermentation process or a batch fermentation process.

The present invention also provides methods of producing an IL-29polypeptide comprising culturing a host cell in a suitable growth mediumin a shake flask to an optical density (OD) of between 5 and 20 at 600nm, inoculating a fermentation vessel with 1 to 5% v/v (e.g., 1% v/v and2% v/v) of shake flask medium containing host cells, culturing the hostcells in a growth medium at a pH of 6.2 to 7.2 (e.g., pH 6.8), where afeed solution is fed into the fermentation vessel before 10 to 20 hours(e.g., 15 hours) elapsed fermentation time (EFT), adding an inducingagent to the fermentation vessel at 20 to 30 hours EFT (e.g., 24 hours),and harvesting the host cells at 48 to 56 hours EFT. In one embodiment,the inducing agent is isopropyl thiogalactopyranoside (IPTG) at 0.5 to 2mM (e.g., 1 mM). In another embodiment, the feed solution comprises acarbohydrate, e.g., glycerol and glucose, and the feed is 10 to 30grams/Liter (g/L) (e.g., 10-20 g/L) of carbohydrate per hour. In anotherembodiment, the glycerol in the feed solution is 40 to 70% v/v glycerol(e.g., 50% w/v) or the glucose is 40 to 70% w/v glucose (e.g., 50% w/v).In further embodiments, the glycerol is about 70% v/v or the glucose isabout 60% w/v.

The present invention also provides methods of producing an IL-29polypeptide comprising seeding a flask with an inoculum comprising an E.coli W3110, ZGold1, or ZGold5 host cells expressing an IL-29 polypeptideselected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, andbiologically active mutants, or an E. coli W3110, ZGold1, or ZGold5 hostcell comprising a pTAP440 or pTAP395 vector, wherein an IL-29polypeptide is expressed, and with growth medium comprising about 5 to 7g/L glycerol or glucose, culturing the inoculum in a growth medium for16 to 20 hours at about 30° C. to about 37° C., transferring thecultured inoculum in growth medium to a batch fermentator at aconcentration 1 to 5% v/v inoculum (e.g., 1 to 2% v/v), fermenting thebatch fermentation at about 37° C. and about pH 6.8 with about 10 to 30g/L (e.g., 10 to 20 g/L) glycerol or glucose, introducing a glucose feedat about 6 to 8 hours EFT of about 5 to 15 grams of glucose or glycerolper liter per hour and continuing until end of a fermentation run,adding IPTG at about 24 hours EFT to final concentration of 0.5 to 2 mM(e.g., 1 mM), fermenting an additional 20 to 30 hours (e.g., 24 hours),harvesting fermentation broth from the fermentor, adding an equal volumeof water to the fermentation broth, and homogenizing and centrifuging tocollect a cell pellet or cell slurry comprising IL-29 protein material.

In another aspect, the present invention provides methods of isolatinginsoluble IL-29 polypeptide comprising a sequence of amino acid residuesselected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, andbiologically active mutants comprising separating water insoluble IL-29polypeptide from a cell pellet or slurry, dissolving the insoluble IL-29material in a chaotropic solvent, diluting the chaotropic solvent andrefolding the IL-29 polypeptide; and isolating the IL-29 polypeptide,wherein the isolated IL-29 polypeptide is capable of being biologicallyactive. In one embodiment of the invention, the isolated IL-29polypeptide is at least 90% pure. In another embodiment, the isolatedIL-29 polypeptide is at least 90% pure and has an endotoxin level ofless than 10 endotoxin units per mg IL-29 polypeptide in a Limulusamoebocyte lysate assay based on USP <85>.

The present invention also provides a method of isolating insolubleIL-29 polypeptide comprising a sequence of amino acid residues selectedfrom the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, andbiologically active mutants comprising separating from a fermentationbroth a cell pellet or cell slurry comprising water insoluble IL-29polypeptide material, homogenizing the cell pellet or cell slurry tocollect inclusion bodies, dissolving the insoluble IL-29 polypeptidematerial in a chaoptropic solvent comprising a guanidine salt, dilutingthe chaotropic solvent by addition of a refolding buffer comprisingarginine salts and a mixture of reducing and oxidizing components,isolating the IL-29 polypeptide by removing unfolded and aggregatedproteins by filtering, and purifying the IL-29 refolded polypeptide on acation exchange column, wherein the isolated and purified IL-29 iscapable of being biologically active.

In another aspect, the present invention provides a method of isolatinginsoluble IL-29 polypeptide comprising a sequence of amino acid residuesselected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, andbiologically active mutants comprising separating from a fermentationbroth a cell pellet or cell slurry comprising water insoluble IL-29material, homogenizing the cell pellet or cell slurry to collectinclusion bodies, dissolving the insoluble IL-29 protein material in achaotropic solvent comprising a guanidine salt, diluting the chaotropicsolvent by addition of a refolding buffer comprising arginine salts anda mixture of reducing and oxidizing components, isolating the IL-29polypeptide by removing unfolded and aggregated proteins by filtering,purifying the IL-29 refolded polypeptide on a cation exchange column,and purifying the IL-29 eluate on a hydrophobic interaction column,wherein the isolated and purified IL-29 polypeptide is capable of beingbiologically active.

In another aspect, the present invention provides a method for isolatinginsoluble IL-29 polypeptide comprising a sequence of amino acid residuesselected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, andbiologically active mutants comprising separating from a fermentationbroth a cell pellet or cell slurry comprising water insoluble IL-29polypeptide material, homogenizing the cell pellet or cell slurry tocollect inclusion bodies, dissolving the insoluble IL-29 polypeptidematerial in a chaotropic solvent comprising about 6 M guanidinehydrochloride, 40 mM dithiothreitol (DTT) for about one hour at roomtemperature, refolding the dissolved inclusion bodies in a solution bydiluting into refolding buffer comprising about 2 mM DTT, 4 mM cystineoxidation-reduction pair at least 20 times, adjusting the pH to about5.5 with about 20% acetic acid and allowing the solution to react for atleast five hours, diluting the solution with about 1+1.4 volumes 25 mMacetate, pH 5.5, filtering the solution, loading the solution on aTosohaas SP-550C resin column equilibrated to pH 5.5 using sodiumacetate buffer, washing the resin column with about 2 M sodium chloride,washing the resin column with about 0.6 M sodium chloride to elute boundIL-29 polypeptide, adding ammonium sulfate to a concentration of about1.5 M to eluate and filtering eluate solution, loading eluate solutiononto a Tosohaas butyl 650-M column equilibrated to 1.5 M ammoniumsulfate, 0.05 sodium chloride in sodium acetate buffer, diluting eluateonto a SP Sepharose HP column equilibrated with sodium acetate buffer,washing column with 20 column volume linear gradient from 0.3 0.7 Msodium chloride, contration the IL-29 protein, and exchanging buffer toformulation buffer using tangential flow ultrafiltration.

The present invention also provides for the covalently attaching apolyethylene glycol (PEG) to a purified IL-29 polypeptide. The PEG canbe attached to the N- or C-terminus of the IL-29 polypeptide. The PEGmay be 20 kDa methoxyPEG-propionaldehyde. The present invention alsoprovides for the purification of mono-PEGylated IL-29.

The present invention also provides for a method of producing an IL-29polypeptide comprising (a) culturing a prokaryotic host cell comprisinga nucleic acid molecule encoding an IL-29 polypeptide operably linked toan inducible promoter in a first growth medium under conditions whereinthe encoded IL-29 polypeptide is expressed in a shake flask to an OD600of 5 to 20; (b) inoculating a fermentation vessel with 1 to 5% v/v ofshake flask medium containing host cells; (c) culturing the host cellsin a second growth medium at a pH of 6.2 to 7.2, wherein a carbohydratefeed solution is fed into the fermentation vessel at 6 to 8 hourselapsed fermentation time; (d) adding an inducing agent to thefermentation vessel at 20 to 30 hours elapsed fermentation time; and (e)harvesting the prokaryotic host cells at 48 to 56 hours elapsedfermentation time. Optionally, the carbohydrate feed solution maycomprise a glycerol or glucose at a concentration of 10 to 30 g/L growthmedium, and a feed rate of 5-15 grams of glycerol or glucose per literper hour. The prokaryotic host cell may be Escherichia coli, such as,for instance, W3110, ZGOLD1, and ZGOLD5. In addition, the prokaryotichost cell, e.g., Escherichia coli, may be OmpT deficient and/or fhuAdeficient. The encoded IL-29 polypeptide may include an amino acidsequence selected from the group of SEQ ID NOs:2, 4, 6, 8, 10 and 12.The inducing agent of step (d) may be isopropyl thiogalactopyranoside,which may be added to the culture at a concentration of 0.5 mM to 2 mM.

The present invention also provides a method of recovering an IL-29polypeptide from a prokaryotic host cell comprising (a) culturing aprokaryotic host cell comprising a nucleic acid molecule encoding anIL-29 polypeptide operably linked to an inducible promoter in growthmedium under conditions wherein the encoded IL-29 polypeptide isexpressed; (b) adding an inducing agent to induce expression of theIL-29 polypeptide; (c) harvesting the prokaryotic host cells; (d) lysingthe prokaryotic host cells; (e) centrifuging the lysed prokaryotic hostcells; (f) recovering the inclusion body pellet; (g) solubilizing theinclusion body pellet in 4-6 M guanidine hydrochloride and 10-50 mMdithiothreitol for 1-2 hours at 15-25° C.; and (h) adding thesolubilized IL-29 polypeptide to a refolding buffer comprising 0.05-0.5%polyethylene glycol, salt, 0.5 M-1.25 M arginine and a mixture ofreduced and oxidized molecules for 1-26 hours at a temperature of 4-30°C. and a pH 7.3-8.5, wherein the solubilized IL-29 polypeptide isrefolded; (i) quenching the refolding reaction by adjusting the pH to5.5-6.5; (j) diluting the quenched refolding solution 1.5- to 10-fold inwater or low ionic strength buffer at pH 5-7; and (k) filtering thequenched, diluted refold solution through filters to remove precipitateor particulates. The prokaryotic host cells of step (d) may be lysed byhomogenization. The lysed prokaryotic host cells of step (e) may becentrifuged by either batch or continuous centrifugation. The IL-29polypeptide of step (h) may be added to the refolding buffer to a finalconcentration of 0.05-3.0 mg/ml. The mixture of reduced and oxidizedmolecules of the refolding buffer of step (h) may be molecules selectedfrom the group of cysteine and cystine, dithiothreitol and cystine,reduced glutathione and oxidized glutathione, and dithiothreitol andoxidized glutathione. The present invention also provides for an IL-29polypeptide produced and/or recovered by methods as described herein.

The present invention also provides a method of purifying an IL-29polypeptide comprising (a) providing the IL-29 polypeptide according tostep (k) of claim 13; (b) loading the filtered solution comprisingrefolded IL-29 polypeptide of step (a) onto a cation exchangechromatography column equilibrated with sodium acetate at pH 5.5; (c)eluting bound IL-29 polypeptide with sodium chloride in sodium acetate,pH 5.5; and (d) adjusting the eluate with ammonium sulfate to 1 Mconcentration, and passing the adjusted IL-29 polypeptide eluate througha 0.45 μm filter. Optionally, the IL-29 polypeptide may elute from thecation exchange column to form a pool at about 0.7 M-0.8 M sodiumchloride after using a linear gradient elution of 0-2M sodium chloride.In another aspect in purifying an IL-29 polypeptide the method mayfurther comprise (e) loading the IL-29 polypeptide of step (d) onto ahydrophobic interaction chromatography column equilibrated with 50 mMsodium acetate, 1.5 M ammonium sulfate, pH 5.5; (f) eluting the IL-29polypeptide with a linear 50 mM sodium acetate, 1.5 M ammonium sulfateto 50 mM sodium acetate with no ammonium sulfate, pH 5.5; (g) dilutingthe eluate about 6-fold with water or low ionic strength buffer andpassing the diluted IL-29 polypeptide eluate through a 0.2 μm or 0.45 μmfilter. Optionally, the IL-29 polypeptide may elute from the hydrophobicinteraction chromatography column at about 0.75 M ammonium sulfate to 0M ammonium sulfate. In another aspect in purifying an IL-29 polypeptidethe method may even further comprise (h) loading the IL-29 polypeptideof step (g) onto a high performance cation exchange chromatographycolumn equilibrated with 50 mM sodium acetate comprising 0-300 mM sodiumchloride, pH 5.5; and (i) eluting the IL-29 polypeptide with a higherconcentration of sodium chloride in 50 mM sodium acetate, pH 5.5, in astep or gradient elution format. Optionally, the IL-29 polypeptide mayelute from the high performance cation exchange chromatography column atabout 0.4 M sodium chloride to 0.6 M sodium chloride after using agradient elution of 300 to 800 mM sodium chloride. The IL-29 polypeptidemay be at least 98% pure by sodium dodecyl sulfate polyacrylamide gelanalysis and aggregates may be less than 0.2% by size exclusion HPLC.The present invention also provides for an IL-29 polypeptide producedand/or recovered and/or purified by methods as described herein.

The present invention also provides a method of concentrating a purifiedIL-29 polypeptide comprising (a) providing a purified IL-29 polypeptideas described herein; (b) adding the IL-29 polypeptide to a tangentialflow filtration plate and frame system comprising one or more 3-10 kDamolecular weight cut-off membrane; (c) applying a transmembrane pressureof 15-25 psi to the system to ultrafilter the solution to a higherconcentration; and (c) filtering the concentrated IL-29 polypeptidethrough a 0.2 μm membrane. The IL-29 polypeptide may be at least 98%pure by sodium dodecyl sulfate polyacrylamide gel analysis andaggregates may be less than 0.2% by size exclusion HPLC. The IL-29polypeptide may have an endotoxin level of less than 10 endotoxin unitsper milligram of IL-29 polypeptide in a Limulus amoebocyte lysate assaybased on USP <85>. The present invention also provides for an IL-29polypeptide produced and/or recovered and/or purified and/orconcentrating by methods as described herein.

The present invention also provides a method of monopegylating an IL-29polypeptide comprising (a) providing 3-5 g/L IL-29 polypeptide in asodium acetate buffer solution; (b) adding 10-20 mM sodiumcyanoborohydride to the solution of step (a); (c) adding a 2-fold molarexcess of derivatized polyethylene glycol to the solution of step (b);and (d) mixing the solution of step (c) for 10-18 hours at 16-20° C.Optionally, the monopegylated IL-29 polypeptide may have at least 99%monopegylated as measured by reversed phase HPLC. The present inventionalso provides for monopegylated IL-29 polypeptides as produced bymethods as described herein.

The present invention also provides a method of purifying monopegylatedIL-29 polypeptide comprising (e) providing monopegylated IL-29polypeptide as described herein; (f) diluting the solution of step (e)2-fold with 50 mM sodium acetate, pH 5.5; (g) filtering the solution ofstep (f) through a 0.2 μm membrane; (h) loading the solution of step (g)onto a high performance cation exchange chromatography columnequilibrated with 50 mM sodium acetate, 200 mM sodium chloride, pH 5.5;(i) eluting monopegylated IL-29 polypeptide from the high performancecation exchange chromatography column with a linear 50 mM sodiumacetate, 500 mM sodium chloride gradient, pH 5.5; (j) adding themonopegylated IL-29 polypeptide to a tangential flow filtration plateand frame system comprising one or more 3-10 kDa molecular weightcut-off membrane; (k) applying a transmembrane pressure of 15-25 psi tothe system to ultrafilter the solution to a higher concentration; (l)using the system to buffer exchange the concentrated IL-29 polypeptideinto an appropriate formulation buffer by diafiltration; and (m)filtering the concentrated monopegylated IL-29 polypeptide through a 0.2μm membrane. The polyethylene glycol may include a 20 kDa or 30 kDamono-methoxyPEG-propionaldehyde. The polyethylene glycol may beN-terminally or C-terminally attached to the IL-29 polypeptide.Optionally, the monopegylated IL-29 polypeptide may have at least 99%monopegylated as measured by reversed phase HPLC. The present inventionalso provides for monopegylated IL-29 polypeptides as produced andpurified by methods as described herein.

IL-29 Polynucleotides and Polypeptides

The human IL-29 gene encodes a mature polypeptide, not including thesignal sequence, of 182 amino acids. The IL-29 sequence as expressedusing a prokaryotic expression system has an N-terminal methionine, andthe nucleotide and corresponding amino acid sequences are shown in SEQID NOs:11 and 12 (referred to herein as IL-29 wildtype sequences),respectively. The nucleotide sequence of SEQ ID NO:11 shows a codonoptimized sequence that is within the scope of the present invention.“IL-29”, “recombinant IL-29”, “recombinant human IL-29”, are usedinterchangeably herein and refer to an IL-29 molecule in general andinclude IL-29 wildtype (SEQ ID NO:12), IL-29C172S (SEQ ID NO:2), IL-29C172S Leucine Insert (SEQ ID NO:4), IL-29 C172S d2-7 (SEQ ID NO:6),IL-29 C1 mutants (SEQ ID NO:8), IL-29 C5 mutants (SEQ ID NO:10),fragments (N-terminal, C-terminal and N- and C-terminal fragments),variants and fusions thereof.

Zcyto21 or IL-29 polypeptides of the present invention also include amutation at the fifth cysteine, C5, of the mature polypeptide. Forexample, C5 from the N-terminus of the polypeptide of SEQ ID NO:12, isthe cysteine at position 172. This fifth cysteine or C5 of IL-29 can bemutated, for example, to any amino acid which will not form a disulfidebond with another cysteine (e.g., serine, alanine, threonine, valine, orasparagine). These IL-29 C5 mutant polypeptides have a disulfide bondpattern of C1 (Cys16 of SEQ ID NO:10)/C3 (Cys113 of SEQ ID NO:10) and C2(Cys50 of SEQ ID NO:10)/C4 (Cys146 of SEQ ID NO:10). IL-29 C5 mutantmolecules of the present invention include polynucleotide molecules asshown in SEQ ID NO:9, including DNA and RNA molecules, that encode IL-29C5 mutant polypeptides as shown in SEQ ID NO:10 (U.S. Patent ApplicationSer. Nos. 60/700,905 and 60/700,951, PCT publication WO 03/066002(Kotenko et al.) and PCT publication WO 02/092762 (Baum et al.)).

The various uses, for example, for an IL-29 molecule of the presentinvention include a use as an antiviral drug (e.g., for the treatment ofhepatitis C, hepatitis B, human immunodeficiency virus) as well as atherapeutic agent for various autoimmune disorders (e.g., multiplesclerosis) and various cancers (e.g., hepatocellular carcinoma, renalcell carcinoma, pancreatic cancer, colon cancer, various B-cellmalignancies), which are more fully disclosed in commonly assigned U.S.Pat. No. 6,927,040, U.S. Pat. No. 7,038,032, WO 04/037995, WO 05/023862,U.S. Patent Publication No. 2005-0244423, U.S. Patent Publication No.2006-012644, U.S. patent application Ser. No. 11/458,945, and U.S.patent application Ser. No. 11/489,894, all of which are hereinincorporated by reference in their entirety.

The present invention also includes biologically active mutants of IL-29C5 cysteine mutants, which provide at least partial antiviral activity,or have therapeutic activity for autoimmune diseases and/or variouscancers. The biologically active mutants of IL-29 C5 cysteine mutants ofthe present invention include N-, C-, and N- and C-terminal deletions ofIL-29, e.g., the polypeptides of SEQ ID NO:10 encoded by thepolynucleotides of SEQ ID NO:9.

N-terminally modified biologically active mutants of IL-29 C5 mutantsinclude, for example, amino acid residues 2-182 of SEQ ID NO:10 which isencoded by nucleotides 4-546 of SEQ ID NO:9; amino acid residues 3-182of SEQ ID NO:10 which is encoded by nucleotides 7-546 of SEQ ID NO:9;amino acid residues 4-182 of SEQ ID NO:10 which is encoded bynucleotides 10-546 of SEQ ID NO:9; amino acid residues 5-182 of SEQ IDNO:10 which is encoded by nucleotides 13-546 of SEQ ID NO:9; amino acidresidues 6-182 of SEQ ID NO:10 which is encoded by nucleotides 16-546 ofSEQ ID NO:9; amino acid residues 7-182 of SEQ ID NO:10 which is encodedby nucleotides 19-546 of SEQ ID NO:9; amino acid residues 8-182 of SEQID NO:10 which is encoded by nucleotides 22-546 of SEQ ID NO:9; aminoacid residues 9-182 of SEQ ID NO:10 which is encoded by nucleotides25-546 of SEQ ID NO:9; amino acid residues 10-182 of SEQ ID NO:10 whichis encoded by nucleotides 28-546 of SEQ ID NO:9; amino acid residues11-182 of SEQ ID NO:10 which is encoded by nucleotides 31-546 of SEQ IDNO:9; amino acid residues 12-182 of SEQ ID NO:10 which is encoded bynucleotides 34-546 of SEQ ID NO:9; amino acid residues 13-182 of SEQ IDNO:10 which is encoded by nucleotides 37-546 of SEQ ID NO:9; amino acidresidues 14-182 of SEQ ID NO:10 which is encoded by nucleotides 40-546of SEQ ID NO:9; amino acid residues 15-182 of SEQ ID NO:10 which isencoded by nucleotides 43-546 of SEQ ID NO:9. The N-terminally modifiedbiologically active mutants of IL-29 C5 mutants of the present inventionmay also include an N-terminal methione if expressed, for instance, inE. coli.

C-terminally modified biologically active mutants of IL-29 C5 mutantsinclude, for example, amino acid residues 1-181 of SEQ ID NO:10 which isencoded by nucleotides 1-543 of SEQ ID NO:9; amino acid residues 1-180of SEQ ID NO:10 which is encoded by nucleotides 1-540 of SEQ ID NO:9;amino acid residues 1-179 of SEQ ID NO:10 which is encoded bynucleotides 1-537 of SEQ ID NO:9; amino acid residues 1-178 of SEQ IDNO:10 which is encoded by nucleotides 1-534 of SEQ ID NO:9; amino acidresidues 1-177 of SEQ ID NO:10 which is encoded by nucleotides 1-531 ofSEQ ID NO:9; amino acid residues 1-176 of SEQ ID NO:10 which is encodedby nucleotides 1-528 of SEQ ID NO:9; amino acid residues 1-175 of SEQ IDNO:10 which is encoded by nucleotides 1-525 of SEQ ID NO:9; amino acidresidues 1-174 of SEQ ID NO:10 which is encoded by nucleotides 1-522 ofSEQ ID NO:9; amino acid residues 1-173 of SEQ ID NO:10 which is encodedby nucleotides 1-519 of SEQ ID NO:9; amino acid residues 1-172 of SEQ IDNO:10 which is encoded by nucleotides 1-516 of SEQ ID NO:9. TheC-terminally modified biologically active mutants of IL-29 C5 mutants ofthe present invention may also include an N-terminal Methione ifexpressed, for instance, in E. coli.

N-terminally and C-terminally modified biologically active mutants ofIL-29 C5 mutants include, for example, amino acid residues 2-182 of SEQID NO:10 which is encoded by nucleotides 4-546 of SEQ ID NO:9; aminoacid residues 2-181 of SEQ ID NO:10 which is encoded by nucleotides4-543 of SEQ ID NO:9; amino acid residues 2-180 of SEQ ID NO:10 which isencoded by nucleotides 4-540 of SEQ ID NO:9; amino acid residues 2-179of SEQ ID NO:10 which is encoded by nucleotides 4-537 of SEQ ID NO:9;amino acid residues 2-178 of SEQ ID NO:10 which is encoded bynucleotides 4-534 of SEQ ID NO:9; amino acid residues 2-177 of SEQ IDNO:10 which is encoded by nucleotides 4-531 of SEQ ID NO:9; amino acidresidues 2-176 of SEQ ID NO:10 which is encoded by nucleotides 4-528 ofSEQ ID NO:9; amino acid residues 2-175 of SEQ ID NO:10 which is encodedby nucleotides 4-525 of SEQ ID NO:9; amino acid residues 2-174 of SEQ IDNO:10 which is encoded by nucleotides 4-522 of SEQ ID NO:9; amino acidresidues 2-173 of SEQ ID NO:10 which is encoded by nucleotides 4-519 ofSEQ ID NO:9; amino acid residues 2-172 of SEQ ID NO:10 which is encodedby nucleotides 4-516 of SEQ ID NO:9; amino acid residues 3-182 of SEQ IDNO:10 which is encoded by nucleotides 7-546 of SEQ ID NO:9; amino acidresidues 3-181 of SEQ ID NO:10 which is encoded by nucleotides 7-543 ofSEQ ID NO:9; amino acid residues 3-180 of SEQ ID NO:10 which is encodedby nucleotides 7-540 of SEQ ID NO:9; amino acid residues 3-179 of SEQ IDNO:10 which is encoded by nucleotides 7-537 of SEQ ID NO:9; amino acidresidues 3-178 of SEQ ID NO:10 which is encoded by nucleotides 7-534 ofSEQ ID NO:9; amino acid residues 3-177 of SEQ ID NO:10 which is encodedby nucleotides 7-531 of SEQ ID NO:9; amino acid residues 3-176 of SEQ IDNO:10 which is encoded by nucleotides 7-528 of SEQ ID NO:9; amino acidresidues 3-175 of SEQ ID NO:10 which is encoded by nucleotides 7-525 ofSEQ ID NO:9; amino acid residues 3-174 of SEQ ID NO:10 which is encodedby nucleotides 7-522 of SEQ ID NO:9; amino acid residues 3-173 of SEQ IDNO:10 which is encoded by nucleotides 7-519 of SEQ ID NO:9; amino acidresidues 3-172 of SEQ ID NO:10 which is encoded by nucleotides 7-516 ofSEQ ID NO:9; amino acid residues 4-182 of SEQ ID NO:10 which is encodedby nucleotides 10-546 of SEQ ID NO:9; amino acid residues 4-181 of SEQID NO:10 which is encoded by nucleotides 10-543 of SEQ ID NO:9; aminoacid residues 4-180 of SEQ ID NO:10 which is encoded by nucleotides10-540 of SEQ ID NO:9; amino acid residues 4-179 of SEQ ID NO:10 whichis encoded by nucleotides 10-537 of SEQ ID NO:9; amino acid residues4-178 of SEQ ID NO:10 which is encoded by nucleotides 10-534 of SEQ IDNO:9; amino acid residues 4-177 of SEQ ID NO:10 which is encoded bynucleotides 10-531 of SEQ ID NO:9; amino acid residues 4-176 of SEQ IDNO:10 which is encoded by nucleotides 10-528 of SEQ ID NO:9; amino acidresidues 4-175 of SEQ ID NO:10 which is encoded by nucleotides 10-525 ofSEQ ID NO:9; amino acid residues 4-174 of SEQ ID NO:10 which is encodedby nucleotides 10-522 of SEQ ID NO:9; amino acid residues 4-173 of SEQID NO:10 which is encoded by nucleotides 10-519 of SEQ ID NO:9; aminoacid residues 4-172 of SEQ ID NO:10 which is encoded by nucleotides10-516 of SEQ ID NO:9; amino acid residues 5-182 of SEQ ID NO:10 whichis encoded by nucleotides 13-546 of SEQ ID NO:9; amino acid residues5-181 of SEQ ID NO:10 which is encoded by nucleotides 13-543 of SEQ IDNO:9; amino acid residues 5-180 of SEQ ID NO:10 which is encoded bynucleotides 13-540 of SEQ ID NO:9; amino acid residues 5-179 of SEQ IDNO:10 which is encoded by nucleotides 13-537 of SEQ ID NO:9; amino acidresidues 5-178 of SEQ ID NO:10 which is encoded by nucleotides 13-534 ofSEQ ID NO:9; amino acid residues 5-177 of SEQ ID NO:10 which is encodedby nucleotides 13-531 of SEQ ID NO:9; amino acid residues 5-176 of SEQID NO:10 which is encoded by nucleotides 13-528 of SEQ ID NO:9; aminoacid residues 5-175 of SEQ ID NO:10 which is encoded by nucleotides13-525 of SEQ ID NO:9; amino acid residues 5-174 of SEQ ID NO:10 whichis encoded by nucleotides 13-522 of SEQ ID NO:9; amino acid residues5-173 of SEQ ID NO:10 which is encoded by nucleotides 13-519 of SEQ IDNO:9; amino acid residues 5-172 of SEQ ID NO:10 which is encoded bynucleotides 13-516 of SEQ ID NO:9; amino acid residues 6-182 of SEQ IDNO:10 which is encoded by nucleotides 16-546 of SEQ ID NO:9; amino acidresidues 6-181 of SEQ ID NO:10 which is encoded by nucleotides 16-543 ofSEQ ID NO:9; amino acid residues 6-180 of SEQ ID NO:10 which is encodedby nucleotides 16-540 of SEQ ID NO:9; amino acid residues 6-179 of SEQID NO:10 which is encoded by nucleotides 16-537 of SEQ ID NO:9; aminoacid residues 6-178 of SEQ ID NO:10 which is encoded by nucleotides16-534 of SEQ ID NO:9; amino acid residues 6-177 of SEQ ID NO:10 whichis encoded by nucleotides 16-531 of SEQ ID NO:9; amino acid residues6-176 of SEQ ID NO:10 which is encoded by nucleotides 16-528 of SEQ IDNO:9; amino acid residues 6-175 of SEQ ID NO:10 which is encoded bynucleotides 16-525 of SEQ ID NO:9; amino acid residues 6-174 of SEQ IDNO:10 which is encoded by nucleotides 16-522 of SEQ ID NO:9; amino acidresidues 6-173 of SEQ ID NO:10 which is encoded by nucleotides 16-519 ofSEQ ID NO:9; amino acid residues 6-172 of SEQ ID NO:10 which is encodedby nucleotides 16-516 of SEQ ID NO:9; amino acid residues 7-182 of SEQID NO:10 which is encoded by nucleotides 19-546 of SEQ ID NO:9; aminoacid residues 7-181 of SEQ ID NO:10 which is encoded by nucleotides19-543 of SEQ ID NO:9; amino acid residues 7-180 of SEQ ID NO:10 whichis encoded by nucleotides 19-540 of SEQ ID NO:9; amino acid residues7-179 of SEQ ID NO:10 which is encoded by nucleotides 19-537 of SEQ IDNO:9; amino acid residues 7-178 of SEQ ID NO:10 which is encoded bynucleotides 19-534 of SEQ ID NO:9; amino acid residues 7-177 of SEQ IDNO:10 which is encoded by nucleotides 19-531 of SEQ ID NO:9; amino acidresidues 7-176 of SEQ ID NO:10 which is encoded by nucleotides 19-528 ofSEQ ID NO:9; amino acid residues 7-175 of SEQ ID NO:10 which is encodedby nucleotides 19-525 of SEQ ID NO:9; amino acid residues 7-174 of SEQID NO:10 which is encoded by nucleotides 19-522 of SEQ ID NO:9; aminoacid residues 7-173 of SEQ ID NO:10 which is encoded by nucleotides19-519 of SEQ ID NO:9; amino acid residues 7-172 of SEQ ID NO:10 whichis encoded by nucleotides 19-516 of SEQ ID NO:9; amino acid residues8-182 of SEQ ID NO:10 which is encoded by nucleotides 22-546 of SEQ IDNO:9; amino acid residues 8-181 of SEQ ID NO:10 which is encoded bynucleotides 22-543 of SEQ ID NO:9; amino acid residues 8-180 of SEQ IDNO:10 which is encoded by nucleotides 22-540 of SEQ ID NO:9; amino acidresidues 8-179 of SEQ ID NO:10 which is encoded by nucleotides 22-537 ofSEQ ID NO:9; amino acid residues 8-178 of SEQ ID NO:10 which is encodedby nucleotides 22-534 of SEQ ID NO:9; amino acid residues 8-177 of SEQID NO:10 which is encoded by nucleotides 22-531 of SEQ ID NO:9; aminoacid residues 8-176 of SEQ ID NO:10 which is encoded by nucleotides22-528 of SEQ ID NO:9; amino acid residues 8-175 of SEQ ID NO:10 whichis encoded by nucleotides 22-525 of SEQ ID NO:9; amino acid residues8-174 of SEQ ID NO:10 which is encoded by nucleotides 22-522 of SEQ IDNO:9; amino acid residues 8-173 of SEQ ID NO:10 which is encoded bynucleotides 22-519 of SEQ ID NO:9; amino acid residues 8-172 of SEQ IDNO:10 which is encoded by nucleotides 22-516 of SEQ ID NO:9; amino acidresidues 9-182 of SEQ ID NO:10 which is encoded by nucleotides 25-546 ofSEQ ID NO:9; amino acid residues 9-181 of SEQ ID NO:10 which is encodedby nucleotides 25-543 of SEQ ID NO:9; amino acid residues 9-180 of SEQID NO:10 which is encoded by nucleotides 25-540 of SEQ ID NO:9; aminoacid residues 9-179 of SEQ ID NO:10 which is encoded by nucleotides25-537 of SEQ ID NO:9; amino acid residues 9-178 of SEQ ID NO:10 whichis encoded by nucleotides 25-534 of SEQ ID NO:9; amino acid residues9-177 of SEQ ID NO:10 which is encoded by nucleotides 25-531 of SEQ IDNO:9; amino acid residues 9-176 of SEQ ID NO:10 which is encoded bynucleotides 25-528 of SEQ ID NO:9; amino acid residues 9-175 of SEQ IDNO:10 which is encoded by nucleotides 25-525 of SEQ ID NO:9; amino acidresidues 9-174 of SEQ ID NO:10 which is encoded by nucleotides 25-522 ofSEQ ID NO:9; amino acid residues 9-173 of SEQ ID NO:10 which is encodedby nucleotides 25-519 of SEQ ID NO:9; amino acid residues 9-172 of SEQID NO:10 which is encoded by nucleotides 25-516 of SEQ ID NO:9; aminoacid residues 10-182 of SEQ ID NO:10 which is encoded by nucleotides28-546 of SEQ ID NO:9; amino acid residues 10-181 of SEQ ID NO:10 whichis encoded by nucleotides 28-543 of SEQ ID NO:9; amino acid residues10-180 of SEQ ID NO:10 which is encoded by nucleotides 28-540 of SEQ IDNO:9; amino acid residues 10-179 of SEQ ID NO:10 which is encoded bynucleotides 28-537 of SEQ ID NO:9; amino acid residues 10-178 of SEQ IDNO:10 which is encoded by nucleotides 28-534 of SEQ ID NO:9; amino acidresidues 10-177 of SEQ ID NO:10 which is encoded by nucleotides 28-531of SEQ ID NO:9; amino acid residues 10-176 of SEQ ID NO:10 which isencoded by nucleotides 28-528 of SEQ ID NO:9; amino acid residues 10-175of SEQ ID NO:10 which is encoded by nucleotides 28-525 of SEQ ID NO:9;amino acid residues 10-174 of SEQ ID NO:10 which is encoded bynucleotides 28-522 of SEQ ID NO:9; amino acid residues 10-173 of SEQ IDNO:10 which is encoded by nucleotides 28-519 of SEQ ID NO:9; amino acidresidues 10-172 of SEQ ID NO:10 which is encoded by nucleotides 28-516of SEQ ID NO:9; amino acid residues 11-182 of SEQ ID NO:10 which isencoded by nucleotides 31-546 of SEQ ID NO:9; amino acid residues 11-181of SEQ ID NO:10 which is encoded by nucleotides 31-543 of SEQ ID NO:9;amino acid residues 11-180 of SEQ ID NO:10 which is encoded bynucleotides 31-540 of SEQ ID NO:9; amino acid residues 11-179 of SEQ IDNO:10 which is encoded by nucleotides 31-537 of SEQ ID NO:9; amino acidresidues 11-178 of SEQ ID NO:10 which is encoded by nucleotides 31-534of SEQ ID NO:9; amino acid residues 11-177 of SEQ ID NO:10 which isencoded by nucleotides 31-531 of SEQ ID NO:9; amino acid residues 11-176of SEQ ID NO:10 which is encoded by nucleotides 31-528 of SEQ ID NO:9;amino acid residues 11-175 of SEQ ID NO:10 which is encoded bynucleotides 31-525 of SEQ ID NO:9; amino acid residues 11-174 of SEQ IDNO:10 which is encoded by nucleotides 31-522 of SEQ ID NO:9; amino acidresidues 11-173 of SEQ ID NO:10 which is encoded by nucleotides 31-519of SEQ ID NO:9; amino acid residues 11-172 of SEQ ID NO:10 which isencoded by nucleotides 31-516 of SEQ ID NO:9; amino acid residues 12-182of SEQ ID NO:10 which is encoded by nucleotides 34-546 of SEQ ID NO:9;amino acid residues 12-181 of SEQ ID NO:10 which is encoded bynucleotides 34-543 of SEQ ID NO:9; amino acid residues 12-180 of SEQ IDNO:10 which is encoded by nucleotides 34-540 of SEQ ID NO:9; amino acidresidues 12-179 of SEQ ID NO:10 which is encoded by nucleotides 34-537of SEQ ID NO:9; amino acid residues 12-178 of SEQ ID NO:10 which isencoded by nucleotides 34-534 of SEQ ID NO:9; amino acid residues 12-177of SEQ ID NO:10 which is encoded by nucleotides 34-531 of SEQ ID NO:9;amino acid residues 12-176 of SEQ ID NO:10 which is encoded bynucleotides 34-528 of SEQ ID NO:9; amino acid residues 12-175 of SEQ IDNO:10 which is encoded by nucleotides 34-525 of SEQ ID NO:9; amino acidresidues 12-174 of SEQ ID NO:10 which is encoded by nucleotides 34-522of SEQ ID NO:9; amino acid residues 12-173 of SEQ ID NO:10 which isencoded by nucleotides 34-519 of SEQ ID NO:9; amino acid residues 12-172of SEQ ID NO:10 which is encoded by nucleotides 34-516 of SEQ ID NO:9;amino acid residues 13-182 of SEQ ID NO:10 which is encoded bynucleotides 37-546 of SEQ ID NO:9; amino acid residues 13-181 of SEQ IDNO:10 which is encoded by nucleotides 37-543 of SEQ ID NO:9; amino acidresidues 13-180 of SEQ ID NO:10 which is encoded by nucleotides 37-540of SEQ ID NO:9; amino acid residues 13-179 of SEQ ID NO:10 which isencoded by nucleotides 37-537 of SEQ ID NO:9; amino acid residues 13-178of SEQ ID NO:10 which is encoded by nucleotides 37-534 of SEQ ID NO:9;amino acid residues 13-177 of SEQ ID NO:10 which is encoded bynucleotides 37-531 of SEQ ID NO:9; amino acid residues 13-176 of SEQ IDNO:10 which is encoded by nucleotides 37-528 of SEQ ID NO:9; amino acidresidues 13-175 of SEQ ID NO:10 which is encoded by nucleotides 37-525of SEQ ID NO:9; amino acid residues 13-174 of SEQ ID NO:10 which isencoded by nucleotides 37-522 of SEQ ID NO:9; amino acid residues 13-173of SEQ ID NO:10 which is encoded by nucleotides 37-519 of SEQ ID NO:9;amino acid residues 13-172 of SEQ ID NO:10 which is encoded bynucleotides 37-516 of SEQ ID NO:9; amino acid residues 14-182 of SEQ IDNO:10 which is encoded by nucleotides 40-546 of SEQ ID NO:9; amino acidresidues 14-181 of SEQ ID NO:10 which is encoded by nucleotides 40-543of SEQ ID NO:9; amino acid residues 14-180 of SEQ ID NO:10 which isencoded by nucleotides 40-540 of SEQ ID NO:9; amino acid residues 14-179of SEQ ID NO:10 which is encoded by nucleotides 40-537 of SEQ ID NO:9;amino acid residues 14-178 of SEQ ID NO:10 which is encoded bynucleotides 40-534 of SEQ ID NO:9; amino acid residues 14-177 of SEQ IDNO:10 which is encoded by nucleotides 40-531 of SEQ ID NO:9; amino acidresidues 14-176 of SEQ ID NO:10 which is encoded by nucleotides 40-528of SEQ ID NO:9; amino acid residues 14-175 of SEQ ID NO:10 which isencoded by nucleotides 40-525 of SEQ ID NO:9; amino acid residues 14-174of SEQ ID NO:10 which is encoded by nucleotides 40-522 of SEQ ID NO:9;amino acid residues 40-173 of SEQ ID NO:10 which is encoded bynucleotides 40-519 of SEQ ID NO:9; amino acid residues 14-172 of SEQ IDNO:10 which is encoded by nucleotides 40-516 of SEQ ID NO:9; amino acidresidues 15-182 of SEQ ID NO:10 which is encoded by nucleotides 43-546of SEQ ID NO:9; amino acid residues 15-181 of SEQ ID NO:10 which isencoded by nucleotides 43-543 of SEQ ID NO:9; amino acid residues 15-180of SEQ ID NO:10 which is encoded by nucleotides 43-540 of SEQ ID NO:9;amino acid residues 15-179 of SEQ ID NO:10 which is encoded bynucleotides 43-537 of SEQ ID NO:9; amino acid residues 15-178 of SEQ IDNO:10 which is encoded by nucleotides 43-534 of SEQ ID NO:9; amino acidresidues 15-177 of SEQ ID NO:10 which is encoded by nucleotides 43-531of SEQ ID NO:9; amino acid residues 15-176 of SEQ ID NO:10 which isencoded by nucleotides 43-528 of SEQ ID NO:9; amino acid residues 15-175of SEQ ID NO:10 which is encoded by nucleotides 43-525 of SEQ ID NO:9;amino acid residues 15-174 of SEQ ID NO:10 which is encoded bynucleotides 43-522 of SEQ ID NO:9; amino acid residues 15-173 of SEQ IDNO:10 which is encoded by nucleotides 43-519 of SEQ ID NO:9; amino acidresidues 15-172 of SEQ ID NO:10 which is encoded by nucleotides 43-516of SEQ ID NO:9; amino acid residues 16-182 of SEQ ID NO:10 which isencoded by nucleotides 46-546 of SEQ ID NO:9; amino acid residues 16-181of SEQ ID NO:10 which is encoded by nucleotides 46-543 of SEQ ID NO:9;amino acid residues 16-180 of SEQ ID NO:10 which is encoded bynucleotides 46-540 of SEQ ID NO:9; amino acid residues 16-179 of SEQ IDNO:10 which is encoded by nucleotides 46-537 of SEQ ID NO:9; amino acidresidues 16-178 of SEQ ID NO:10 which is encoded by nucleotides 46-534of SEQ ID NO:9; amino acid residues 16-177 of SEQ ID NO:10 which isencoded by nucleotides 46-531 of SEQ ID NO:9; amino acid residues 16-176of SEQ ID NO:10 which is encoded by nucleotides 46-528 of SEQ ID NO:9;amino acid residues 16-175 of SEQ ID NO:10 which is encoded bynucleotides 46-525 of SEQ ID NO:9; amino acid residues 16-174 of SEQ IDNO:10 which is encoded by nucleotides 46-522 of SEQ ID NO:9; amino acidresidues 16-173 of SEQ ID NO:10 which is encoded by nucleotides 46-519of SEQ ID NO:9; and amino acid residues 16-172 of SEQ ID NO:10 which isencoded by nucleotides 46-516 of SEQ ID NO:9. The N-terminally andC-terminally modified biologically active mutants of IL-29 C5 mutants ofthe present invention may also include an N-terminal Methione ifexpressed, for instance, in E. coli.

In addition to the IL-29 C5 mutants, the present invention also includesIL-29 polypeptides comprising a mutation at the first cysteine position,C1, of the mature polypeptide. For example, C1 from the N-terminus ofthe polypeptide of SEQ ID NO:12, is the cysteine at position 16. TheseIL-29 C1 mutant polypeptides have a predicted disulfide bond pattern ofC2 (Cys50 of SEQ ID NO:8)/C4 (Cys146 of SEQ ID NO:8) and C3 (Cys113 ofSEQ ID NO:8)/C5 (Cys172 of SEQ ID NO:8). IL-29 C1 mutant molecules ofthe present invention include polynucleotide molecules as shown in SEQID NO:7, including DNA and RNA molecules, that encode IL-29 C1 mutantpolypeptides as shown in SEQ ID NO:8.

The present invention also includes biologically active mutants of IL-29C1 cysteine mutants which provide at least partial antiviral activity,or have therapeutic activity for autoimmune diseases and/or variouscancers. The biologically active mutants of IL-29 C1 cysteine mutants ofthe present invention include N-, C-, and N- and C-terminal deletions ofIL-29, e.g., the polypeptides of SEQ ID NO:8 encoded by thepolynucleotides of SEQ ID NO:7.

N-terminally modified biologically active mutants of IL-29 C1 mutantsinclude, for example, amino acid residues 2-182 of SEQ ID NO:8 which isencoded by nucleotides 4-546 of SEQ ID NO:7; amino acid residues 3-182of SEQ ID NO:8 which is encoded by nucleotides 7-546 of SEQ ID NO:7;amino acid residues 4-182 of SEQ ID NO:8 which is encoded by nucleotides10-546 of SEQ ID NO:7; amino acid residues 5-182 of SEQ ID NO:8 which isencoded by nucleotides 13-546 of SEQ ID NO:7; amino acid residues 6-182of SEQ ID NO:8 which is encoded by nucleotides 16-546 of SEQ ID NO:7;amino acid residues 7-182 of SEQ ID NO:8 which is encoded by nucleotides19-546 of SEQ ID NO:7; amino acid residues 8-182 of SEQ ID NO:8 which isencoded by nucleotides 22-546 of SEQ ID NO:7; amino acid residues 9-182of SEQ ID NO:8 which is encoded by nucleotides 25-546 of SEQ ID NO:7;amino acid residues 10-182 of SEQ ID NO:8 which is encoded bynucleotides 28-546 of SEQ ID NO:7; amino acid residues 11-182 of SEQ IDNO:8 which is encoded by nucleotides 31-546 of SEQ ID NO:7; amino acidresidues 12-182 of SEQ ID NO:8 which is encoded by nucleotides 34-182 ofSEQ ID NO:7; amino acid residues 13-182 of SEQ ID NO:8 which is encodedby nucleotides 37-546 of SEQ ID NO:7; amino acid residues 14-182 of SEQID NO:8 which is encoded by nucleotides 40-546 of SEQ ID NO:7; aminoacid residues 15-182 of SEQ ID NO:8 which is encoded by nucleotides43-546 of SEQ ID NO:7; and amino acid residues 16-182 of SEQ ID NO:8which is encoded by nucleotides 46-546 of SEQ ID NO:7. The N-terminallymodified biologically active mutants of IL-29 C1 mutants of the presentinvention may also include an N-terminal Methione if expressed, forinstance, in E. coli.

C-terminally modified biologically active mutants of IL-29 C1 mutantsinclude, for example, amino acid residues 1-181 of SEQ ID NO:8 which isencoded by nucleotides 1-543 of SEQ ID NO:7; amino acid residues 1-180of SEQ ID NO:8 which is encoded by nucleotides 1-540 of SEQ ID NO:7;amino acid residues 1-179 of SEQ ID NO:8 which is encoded by nucleotides1-537 of SEQ ID NO:7; amino acid residues 1-178 of SEQ ID NO:8 which isencoded by nucleotides 1-534 of SEQ ID NO:7; amino acid residues 1-177of SEQ ID NO:8 which is encoded by nucleotides 1-531 of SEQ ID NO:7;amino acid residues 1-176 of SEQ ID NO:8 which is encoded by nucleotides1-528 of SEQ ID NO:7; amino acid residues 1-175 of SEQ ID NO:8 which isencoded by nucleotides 1-525 of SEQ ID NO:7; amino acid residues 1-174of SEQ ID NO:8 which is encoded by nucleotides 1-522 of SEQ ID NO:7;amino acid residues 1-173 of SEQ ID NO:8 which is encoded by nucleotides1-519 of SEQ ID NO:7; and amino acid residues 1-172 of SEQ ID NO:8 whichis encoded by nucleotides 1-516 of SEQ ID NO:7. The C-terminallymodified biologically active mutants of IL-29 C1 mutants of the presentinvention may also include an N-terminal methione if expressed, forinstance, in E. coli.

N-terminally and C-terminally modified biologically active mutants ofIL-29 C1 mutants include, for example, amino acid residues 2-181 of SEQID NO:8 which is encoded by nucleotides 4-543 of SEQ ID NO:7; amino acidresidues 2-180 of SEQ ID NO:8 which is encoded by nucleotides 4-540 ofSEQ ID NO:7; amino acid residues 2-179 of SEQ ID NO:8 which is encodedby nucleotides 4-537 of SEQ ID NO:7; amino acid residues 2-178 of SEQ IDNO:8 which is encoded by nucleotides 4-534 of SEQ ID NO:7; amino acidresidues 2-177 of SEQ ID NO:8 which is encoded by nucleotides 4-531 ofSEQ ID NO:7; amino acid residues 2-176 of SEQ ID NO:8 which is encodedby nucleotides 4-528 of SEQ ID NO:7; amino acid residues 2-175 of SEQ IDNO:8 which is encoded by nucleotides 4-525 of SEQ ID NO:7; amino acidresidues 2-174 of SEQ ID NO:8 which is encoded by nucleotides 4-522 ofSEQ ID NO:7; amino acid residues 2-173 of SEQ ID NO:8 which is encodedby nucleotides 4-519 of SEQ ID NO:7; amino acid residues 2-172 of SEQ IDNO:8 which is encoded by nucleotides 4-516 of SEQ ID NO:7; amino acidresidues 3-181 of SEQ ID NO:8 which is encoded by nucleotides 7-543 ofSEQ ID NO:7; amino acid residues 3-180 of SEQ ID NO:8 which is encodedby nucleotides 7-540 of SEQ ID NO:7; amino acid residues 3-179 of SEQ IDNO:8 which is encoded by nucleotides 7-537 of SEQ ID NO:7; amino acidresidues 3-178 of SEQ ID NO:8 which is encoded by nucleotides 7-534 ofSEQ ID NO:7; amino acid residues 3-177 of SEQ ID NO:8 which is encodedby nucleotides 7-531 of SEQ ID NO:7; amino acid residues 3-176 of SEQ IDNO:8 which is encoded by nucleotides 7-528 of SEQ ID NO:7; amino acidresidues 3-175 of SEQ ID NO:8 which is encoded by nucleotides 7-525 ofSEQ ID NO:7; amino acid residues 3-174 of SEQ ID NO:8 which is encodedby nucleotides 7-522 of SEQ ID NO:7; amino acid residues 3-173 of SEQ IDNO:8 which is encoded by nucleotides 7-519 of SEQ ID NO:7; amino acidresidues 3-172 of SEQ ID NO:8 which is encoded by nucleotides 7-516 ofSEQ ID NO:7; amino acid residues 4-181 of SEQ ID NO:8 which is encodedby nucleotides 10-543 of SEQ ID NO:7; amino acid residues 4-180 of SEQID NO:8 which is encoded by nucleotides 10-540 of SEQ ID NO:7; aminoacid residues 4-179 of SEQ ID NO:8 which is encoded by nucleotides10-537 of SEQ ID NO:7; amino acid residues 4-178 of SEQ ID NO:8 which isencoded by nucleotides 10-534 of SEQ ID NO:7; amino acid residues 4-177of SEQ ID NO:8 which is encoded by nucleotides 10-531 of SEQ ID NO:7;amino acid residues 4-176 of SEQ ID NO:8 which is encoded by nucleotides10-528 of SEQ ID NO:7; amino acid residues 4-175 of SEQ ID NO:8 which isencoded by nucleotides 10-525 of SEQ ID NO:7; amino acid residues 4-174of SEQ ID NO:8 which is encoded by nucleotides 10-522 of SEQ ID NO:7;amino acid residues 4-173 of SEQ ID NO:8 which is encoded by nucleotides10-519 of SEQ ID NO:7; amino acid residues 4-172 of SEQ ID NO:8 which isencoded by nucleotides 10-516 of SEQ ID NO:7; amino acid residues 5-181of SEQ ID NO:8 which is encoded by nucleotides 13-543 of SEQ ID NO:7;amino acid residues 5-180 of SEQ ID NO:8 which is encoded by nucleotides13-540 of SEQ ID NO:7; amino acid residues 5-179 of SEQ ID NO:8 which isencoded by nucleotides 13-537 of SEQ ID NO:7; amino acid residues 5-178of SEQ ID NO:8 which is encoded by nucleotides 13-534 of SEQ ID NO:7;amino acid residues 5-177 of SEQ ID NO:8 which is encoded by nucleotides13-531 of SEQ ID NO:7; amino acid residues 5-176 of SEQ ID NO:8 which isencoded by nucleotides 13-528 of SEQ ID NO:7; amino acid residues 5-175of SEQ ID NO:8 which is encoded by nucleotides 13-525 of SEQ ID NO:7;amino acid residues 5-174 of SEQ ID NO:8 which is encoded by nucleotides13-522 of SEQ ID NO:7; amino acid residues 5-173 of SEQ ID NO:8 which isencoded by nucleotides 13-519 of SEQ ID NO:7; amino acid residues 5-172of SEQ ID NO:8 which is encoded by nucleotides 13-516 of SEQ ID NO:7;amino acid residues 6-181 of SEQ ID NO:8 which is encoded by nucleotides16-543 of SEQ ID NO:7; amino acid residues 6-180 of SEQ ID NO:8 which isencoded by nucleotides 16-540 of SEQ ID NO:7; amino acid residues 6-179of SEQ ID NO:8 which is encoded by nucleotides 16-537 of SEQ ID NO:7;amino acid residues 6-178 of SEQ ID NO:8 which is encoded by nucleotides16-534 of SEQ ID NO:7; amino acid residues 6-177 of SEQ ID NO:8 which isencoded by nucleotides 16-531 of SEQ ID NO:7; amino acid residues 6-176of SEQ ID NO:8 which is encoded by nucleotides 16-528 of SEQ ID NO:7;amino acid residues 6-175 of SEQ ID NO:8 which is encoded by nucleotides16-525 of SEQ ID NO:7; amino acid residues 6-174 of SEQ ID NO:8 which isencoded by nucleotides 16-522 of SEQ ID NO:7; amino acid residues 6-173of SEQ ID NO:8 which is encoded by nucleotides 16-519 of SEQ ID NO:7;amino acid residues 6-172 of SEQ ID NO:8 which is encoded by nucleotides16-516 of SEQ ID NO:7; amino acid residues 7-181 of SEQ ID NO:8 which isencoded by nucleotides 19-543 of SEQ ID NO:7; amino acid residues 7-180of SEQ ID NO:8 which is encoded by nucleotides 19-540 of SEQ ID NO:7;amino acid residues 7-179 of SEQ ID NO:8 which is encoded by nucleotides19-537 of SEQ ID NO:7; amino acid residues 7-178 of SEQ ID NO:8 which isencoded by nucleotides 19-534 of SEQ ID NO:7; amino acid residues 7-177of SEQ ID NO:8 which is encoded by nucleotides 19-531 of SEQ ID NO:7;amino acid residues 7-176 of SEQ ID NO:8 which is encoded by nucleotides19-528 of SEQ ID NO:7; amino acid residues 7-175 of SEQ ID NO:8 which isencoded by nucleotides 19-525 of SEQ ID NO:7; amino acid residues 7-174of SEQ ID NO:8 which is encoded by nucleotides 19-522 of SEQ ID NO:7;amino acid residues 7-173 of SEQ ID NO:8 which is encoded by nucleotides19-519 of SEQ ID NO:7; amino acid residues 7-172 of SEQ ID NO:8 which isencoded by nucleotides 19-516 of SEQ ID NO:7; amino acid residues 8-181of SEQ ID NO:8 which is encoded by nucleotides 22-543 of SEQ ID NO:7;amino acid residues 8-180 of SEQ ID NO:8 which is encoded by nucleotides22-540 of SEQ ID NO:7; amino acid residues 8-179 of SEQ ID NO:8 which isencoded by nucleotides 22-537 of SEQ ID NO:7; amino acid residues 8-178of SEQ ID NO:8 which is encoded by nucleotides 22-534 of SEQ ID NO:7;amino acid residues 8-177 of SEQ ID NO:8 which is encoded by nucleotides22-531 of SEQ ID NO:7; amino acid residues 8-176 of SEQ ID NO:8 which isencoded by nucleotides 22-528 of SEQ ID NO:7; amino acid residues 8-175of SEQ ID NO:8 which is encoded by nucleotides 22-525 of SEQ ID NO:7;amino acid residues 8-174 of SEQ ID NO:8 which is encoded by nucleotides22-522 of SEQ ID NO:7; amino acid residues 8-173 of SEQ ID NO:8 which isencoded by nucleotides 22-519 of SEQ ID NO:7; amino acid residues 8-172of SEQ ID NO:8 which is encoded by nucleotides 22-516 of SEQ ID NO:7;amino acid residues 9-181 of SEQ ID NO:8 which is encoded by nucleotides25-543 of SEQ ID NO:7; amino acid residues 9-180 of SEQ ID NO:8 which isencoded by nucleotides 25-540 of SEQ ID NO:7; amino acid residues 9-179of SEQ ID NO:8 which is encoded by nucleotides 25-537 of SEQ ID NO:7;amino acid residues 9-178 of SEQ ID NO:8 which is encoded by nucleotides25-534 of SEQ ID NO:7; amino acid residues 9-177 of SEQ ID NO:8 which isencoded by nucleotides 25-531 of SEQ ID NO:7; amino acid residues 9-176of SEQ ID NO:8 which is encoded by nucleotides 25-528 of SEQ ID NO:7;amino acid residues 9-175 of SEQ ID NO:8 which is encoded by nucleotides25-525 of SEQ ID NO:7; amino acid residues 9-174 of SEQ ID NO:8 which isencoded by nucleotides 25-522 of SEQ ID NO:7; amino acid residues 9-173of SEQ ID NO:8 which is encoded by nucleotides 25-519 of SEQ ID NO:7;amino acid residues 9-172 of SEQ ID NO:8 which is encoded by nucleotides25-516 of SEQ ID NO:7; amino acid residues 10-181 of SEQ ID NO:8 whichis encoded by nucleotides 28-543 of SEQ ID NO:7; amino acid residues10-180 of SEQ ID NO:8 which is encoded by nucleotides 28-540 of SEQ IDNO:7; amino acid residues 10-179 of SEQ ID NO:8 which is encoded bynucleotides 28-537 of SEQ ID NO:7; amino acid residues 10-178 of SEQ IDNO:8 which is encoded by nucleotides 28-534 of SEQ ID NO:7; amino acidresidues 10-177 of SEQ ID NO:8 which is encoded by nucleotides 28-531 ofSEQ ID NO:7; amino acid residues 10-176 of SEQ ID NO:8 which is encodedby nucleotides 28-528 of SEQ ID NO:7; amino acid residues 10-175 of SEQID NO:8 which is encoded by nucleotides 28-525 of SEQ ID NO:7; aminoacid residues 10-174 of SEQ ID NO:8 which is encoded by nucleotides28-522 of SEQ ID NO:7; amino acid residues 10-173 of SEQ ID NO:8 whichis encoded by nucleotides 28-519 of SEQ ID NO:7; amino acid residues10-172 of SEQ ID NO:8 which is encoded by nucleotides 28-516 of SEQ IDNO:7; amino acid residues 11-181 of SEQ ID NO:8 which is encoded bynucleotides 31-543 of SEQ ID NO:7; amino acid residues 11-180 of SEQ IDNO:8 which is encoded by nucleotides 31-540 of SEQ ID NO:7; amino acidresidues 11-179 of SEQ ID NO:8 which is encoded by nucleotides 31-537 ofSEQ ID NO:7; amino acid residues 11-178 of SEQ ID NO:8 which is encodedby nucleotides 31-534 of SEQ ID NO:7; amino acid residues 11-177 of SEQID NO:8 which is encoded by nucleotides 31-531 of SEQ ID NO:7; aminoacid residues 11-176 of SEQ ID NO:8 which is encoded by nucleotides31-528 of SEQ ID NO:7; amino acid residues 11-175 of SEQ ID NO:8 whichis encoded by nucleotides 31-525 of SEQ ID NO:7; amino acid residues11-174 of SEQ ID NO:8 which is encoded by nucleotides 31-522 of SEQ IDNO:7; amino acid residues 11-173 of SEQ ID NO:8 which is encoded bynucleotides 31-519 of SEQ ID NO:7; amino acid residues 11-172 of SEQ IDNO:8 which is encoded by nucleotides 31-516 of SEQ ID NO:7; amino acidresidues 12-181 of SEQ ID NO:8 which is encoded by nucleotides 34-543 ofSEQ ID NO:7; amino acid residues 12-180 of SEQ ID NO:8 which is encodedby nucleotides 34-540 of SEQ ID NO:7; amino acid residues 12-179 of SEQID NO:8 which is encoded by nucleotides 34-537 of SEQ ID NO:7; aminoacid residues 12-178 of SEQ ID NO:8 which is encoded by nucleotides34-534 of SEQ ID NO:7; amino acid residues 12-177 of SEQ ID NO:8 whichis encoded by nucleotides 34-531 of SEQ ID NO:7; amino acid residues12-176 of SEQ ID NO:8 which is encoded by nucleotides 34-528 of SEQ IDNO:7; amino acid residues 12-175 of SEQ ID NO:8 which is encoded bynucleotides 34-525 of SEQ ID NO:7; amino acid residues 12-174 of SEQ IDNO:8 which is encoded by nucleotides 34-522 of SEQ ID NO:7; amino acidresidues 12-173 of SEQ ID NO:8 which is encoded by nucleotides 34-519 ofSEQ ID NO:7; amino acid residues 12-172 of SEQ ID NO:8 which is encodedby nucleotides 34-516 of SEQ ID NO:7; amino acid residues 13-181 of SEQID NO:8 which is encoded by nucleotides 37-543 of SEQ ID NO:7; aminoacid residues 13-180 of SEQ ID NO:8 which is encoded by nucleotides37-540 of SEQ ID NO:7; amino acid residues 13-179 of SEQ ID NO:8 whichis encoded by nucleotides 37-537 of SEQ ID NO:7; amino acid residues13-178 of SEQ ID NO:8 which is encoded by nucleotides 37-534 of SEQ IDNO:7; amino acid residues 13-177 of SEQ ID NO:8 which is encoded bynucleotides 37-531 of SEQ ID NO:7; amino acid residues 13-176 of SEQ IDNO:8 which is encoded by nucleotides 37-528 of SEQ ID NO:7; amino acidresidues 13-175 of SEQ ID NO:8 which is encoded by nucleotides 37-525 ofSEQ ID NO:7; amino acid residues 13-174 of SEQ ID NO:8 which is encodedby nucleotides 37-522 of SEQ ID NO:7; amino acid residues 13-173 of SEQID NO:8 which is encoded by nucleotides 37-519 of SEQ ID NO:7; aminoacid residues 13-172 of SEQ ID NO:8 which is encoded by nucleotides37-516 of SEQ ID NO:7; amino acid residues 14-181 of SEQ ID NO:8 whichis encoded by nucleotides 40-543 of SEQ ID NO:7; amino acid residues14-180 of SEQ ID NO:8 which is encoded by nucleotides 40-540 of SEQ IDNO:7; amino acid residues 14-179 of SEQ ID NO:8 which is encoded bynucleotides 40-537 of SEQ ID NO:7; amino acid residues 14-178 of SEQ IDNO:8 which is encoded by nucleotides 40-534 of SEQ ID NO:7; amino acidresidues 14-177 of SEQ ID NO:8 which is encoded by nucleotides 40-531 ofSEQ ID NO:7; amino acid residues 14-176 of SEQ ID NO:8 which is encodedby nucleotides 40-528 of SEQ ID NO:7; amino acid residues 14-175 of SEQID NO:8 which is encoded by nucleotides 40-525 of SEQ ID NO:7; aminoacid residues 14-174 of SEQ ID NO:8 which is encoded by nucleotides40-522 of SEQ ID NO:7; amino acid residues 14-173 of SEQ ID NO:8 whichis encoded by nucleotides 40-519 of SEQ ID NO:7; amino acid residues14-172 of SEQ ID NO:8 which is encoded by nucleotides 40-516 of SEQ IDNO:7; amino acid residues 15-181 of SEQ ID NO:8 which is encoded bynucleotides 43-543 of SEQ ID NO:7; amino acid residues 15-180 of SEQ IDNO:8 which is encoded by nucleotides 43-540 of SEQ ID NO:7; amino acidresidues 15-179 of SEQ ID NO:8 which is encoded by nucleotides 43-537 ofSEQ ID NO:7; amino acid residues 15-178 of SEQ ID NO:8 which is encodedby nucleotides 43-534 of SEQ ID NO:7; amino acid residues 15-177 of SEQID NO:8 which is encoded by nucleotides 43-531 of SEQ ID NO:7; aminoacid residues 15-176 of SEQ ID NO:8 which is encoded by nucleotides43-528 of SEQ ID NO:7; amino acid residues 15-175 of SEQ ID NO:8 whichis encoded by nucleotides 43-525 of SEQ ID NO:7; amino acid residues15-174 of SEQ ID NO:8 which is encoded by nucleotides 43-522 of SEQ IDNO:7; amino acid residues 15-173 of SEQ ID NO:8 which is encoded bynucleotides 43-519 of SEQ ID NO:7; amino acid residues 15-172 of SEQ IDNO:8 which is encoded by nucleotides 43-516 of SEQ ID NO:7; amino acidresidues 16-181 of SEQ ID NO:8 which is encoded by nucleotides 46-543 ofSEQ ID NO:7; amino acid residues 16-180 of SEQ ID NO:8 which is encodedby nucleotides 46-540 of SEQ ID NO:7; amino acid residues 16-179 of SEQID NO:8 which is encoded by nucleotides 46-537 of SEQ ID NO:7; aminoacid residues 16-178 of SEQ ID NO:8 which is encoded by nucleotides46-534 of SEQ ID NO:7; amino acid residues 16-177 of SEQ ID NO:8 whichis encoded by nucleotides 46-531 of SEQ ID NO:7; amino acid residues16-176 of SEQ ID NO:8 which is encoded by nucleotides 46-528 of SEQ IDNO:7; amino acid residues 16-175 of SEQ ID NO:8 which is encoded bynucleotides 46-525 of SEQ ID NO:7; amino acid residues 16-174 of SEQ IDNO:8 which is encoded by nucleotides 46-522 of SEQ ID NO:7; amino acidresidues 16-173 of SEQ ID NO:8 which is encoded by nucleotides 46-519 ofSEQ ID NO:7; and amino acid residues 16-172 of SEQ ID NO:8 which isencoded by nucleotides 46-516 of SEQ ID NO:7. The N-terminally andC-terminally modified biologically active mutants of IL-29 C1 mutants ofthe present invention may also include an N-terminal methione ifexpressed, for instance, in E. coli.

The IL-29 polypeptides of the present invention include, for example,SEQ ID NOs:2, 4, 6, 8, 10 and 12, which are encoded by IL-29polynucleotide molecules as shown in SEQ ID NOs:1, 3, 5, 7, 9 and 11,respectively, fragments, mutants (including biologically activeN-terminal, C-terminal and N- and C-terminal mutants), variants andfusions thereof.

Expression of Recombinant IL-29

The present invention provides expression vectors and methods forproducing and purifying recombinant IL-29 protein from a prokaryotichost. IL-29 was previously designated zcyto21 (IL-29 and zcyto21 areused interchangeably herein), and is fully described in commonlyassigned U.S. Pat. No. 6,927,040, U.S. Pat. No. 7,038,032, WO 04/037995,WO 05/023862, U.S. Patent Publication No. 2005-0244423, U.S. PatentPublication No. 2006-012644, U.S. patent application Ser. No.11/458,945, and U.S. patent application Ser. No. 11/489,894, all ofwhich are herein incorporated by reference in their entirety. Inparticular, the expression vectors and methods of the present inventioncomprise an E. coli expression system for the large scale production ofIL-29 utilizing an IL-29 coding sequence with specific changes innucleotides in order to optimize codons and mRNA secondary structure fortranslation in E. coli. Using the expression vectors and growthconditions as described herein significantly improved the yield ofrecombinant protein recovered from the bacteria. In another embodiment,to facilitate the development of high cell density fed-batchfermentation, another E. coli strain, W3110, was selected as a host forthe large scale production of IL-29. This host strain is non-pathogenicand can grow to high cell density in minimally defined fermentationmedia.

The present invention also provides methods for recovering recombinantIL-29 protein from a prokaryotic host when the IL-29 protein isexpressed by the host and found within the host cell as anunglycosylated, insoluble inclusion body. When the prokaryotic cell islysed to isolate the inclusion bodies (also called retractile bodies),the inclusion bodies are aggregates of IL-29. Therefore, the inclusionbodies must be disassociated and dissolved to isolate the IL-29 protein,and generally this requires the use of a denaturing chaotropic solvent,resulting in recovering a polypeptide that must be refolded to havesignificant biological activity. Once the IL-29 protein is refolded, theprotein must be captured and purified. Thus, the present inventionprovides for methods for isolating insoluble IL-29 protein fromprokaryotic cells, dissolving the insoluble IL-29 protein material in achaotropic solvent, diluting the chaotropic solvent in such a mannerthat the IL-29 protein is refolded and isolated. The present inventionalso includes methods for capturing the renatured IL-29 protein from thedilute refold buffer using cation exchange chromatography, and purifyingthe refolded IL-29 protein using hydrophobic interaction chromatography(“HIC”). Further purification is achieved using high performance cationexchange chromatography to remove charged variants from the recombinantIL-29 solution.

The IL-29 DNA coding sequence as used herein comprises the mature humangene, i.e., no signal sequence. The DNA sequence was synthesized toreflect E. coli codon bias, and a methionine was added to the N-terminusof the mature protein for translation initiation.

An optimal E. coli production host should 1) be non-pathogenic; 2)express the target protein well; 3) maintain stability of the expressionvector; and 4) grow well in defined, minimal fermentation media. E. colistrain W3110, for example, can be used as the host for production ofrecombinant protein because it fulfills these requirements. W3110 is aprototrophic derivative of K-12. This strain was isolated in early 1950sby Dr Joshua Lederberg and his research team at the University ofWisconsin. Like other K-12 derivatives, E. coli strain W3110 does notsurvive in non-sterile water, soil, or sewage (Smith H W, Infect Dis.1978 May; 137(5):655-660; Bogosian G. et al., Adv Appl Microbiol. 1991;36:87-131; Bogosian G. et al., Appl Environ Microbiol. 1996 November;62(11):4114-20; Heitkamp M. A. et al., J Ind Microbiol. 1993 July;11(4):243-52; Bogosian G. et al., J Ind Microbiol. 1993 July;11(4):235-41; Bogosian G. et al., J Ind Microbiol. 1992 January;9(1):27-36). Furthermore, this strain is unable to adhere to mammalianintestinal cells and does not colonize the mammalian intestinal tract.Based on these findings, W3110 is considered non-pathogenic and unlikelyto survive in mammalian tissues and cause disease. Additionally, W3110has been used extensively as a host for protein production (Kane J. F.et al., Trends Biotechnol., 6:95-101; and Kane J. F. et al, In: Surfacereactive peptides and polymers: discovery and commercialization (C SSikes and A P Wheeler, eds.) American Chemical Society Books,Washington, D.C.) and grows to very high density in a fed-batchfermentation process.

OmpT is a periplasmic endopeptidase that cleaves specifically betweentwo consecutive basic residues and is active under denaturing conditionssuch as 8 M urea and 6M guanidine-HCl (White C. B. et al., J Biol Chem.,1995 Jun. 2; 270(22):12990-4; and Dekker N. et al., Biochemistry. 2001Feb. 13; 40(6):1694-701). OmpT has been associated with the degradationof recombinant proteins expressed as inclusion bodies in E. coli. WhileW3110 is a robust host for fermentation and expression of the protein,it is not ideal for downstream processing of IL-29. Once the cells arelysed, the OmpT protease may cleave the recombinantly-produced protein.

E. coli is susceptible to infection by T-odd bacteriophage which mayresult in slowed growth or failed fermentations (Ogata S. et al.,Uirusu. 2000 June; 50(1):17-26). This can have serious economicconsequences. The ferrichrome-iron receptor encoded by the fhuA gene ofE. coli K-12 is a multifunctional outer membrane receptor required forthe binding and uptake of ferrichrome and serves as the attachment sitefor T-odd bacteriophage. Antibodies against the carboxyl terminus of thefhuA gene product can prevent infection by bacteriophage T5 (Moeck G. S.et al., J Bacteriol. 1995 November; 177(21):6118-25).

In order to streamline the process for production of IL-29, the ompT andfhuA genes have been removed from the host strain W3110 by homologousrecombination (Datsenko K A, Wanner B L. One-step inactivation ofchromosomal genes in Escherichia coli K-12 using PCR products. Proc NatlAcad Sci USA. 2000 Jun. 6; 97(12):6640-5; Murphy K C, Campellone K G,Poteete A R. PCR-mediated gene replacement in Escherichia coli. Gene.2000 Apr. 4; 246(1-2):321-30; and Yu D, Ellis H M, Lee E C, Jenkins N A,Copeland N G, Court D L. An efficient recombination system forchromosome engineering in Escherichia coli. Proc Natl Acad Sci USA. 2000May 23; 97(11):5978-83.). This newly developed host strain is known asZGOLD5 (more fully described below).

Exemplary production strains of the present invention consist of variousE. coli host strains carrying different expression vectors. For example,E. coli W3110 stably maintains the kanamycin-selectable plasmid forIL-29 expression. W3110 has the genotype F− IN(rrnD⁻ rrnE)1 lambda⁻.ZGOLD1 [F− IN(rrnD⁻ rrnE)1 lambda⁻ ΔompT::tet] is an ΔompT mutantderived from W3110. ZGOLD5 [F− IN(rrnD⁻ rrnE)1 lambda⁻ ΔompT::tetΔfhuA::Cm] is a ΔfhuA mutant derived from ZGOLD1 (W3110). F⁻ denoteslack of the endogenous E. coli F plasmid (New England Biolabs 2005-6Catalog, page 270). IN(rrnD⁻ rrnE)1 denotes an inversion at thechromosomal loci containing the rrnD and rrnE operons (Hill C W and GrayJ W, Genetics., 1988 August; 119(4):771-778). The number 1 indicatesthat this is the first reported allele of this inversion. Thischromosomal rearrangement is predicted to have no effect on productionof IL-29. rph1 is a 1 bp deletion that results in a frame shift over thelast 15 codons in RNase PH, the protein that removes nucleotides fromthe 3′ ends of tRNA precursors. This lesion exerts a polar effect on theadjacent pyrE gene, which encodes orotate phosphoribosyltransferase.This leads to starvation for pyrimidine on minimal medium. This partialauxotrophy, however, can be readily compensated for by supplementing themedia. ilvG is a gene that codes for a subunit of acetolactate synthaseII and acetohydroxybutanoate synthase II. These enzymes are involved invaline/leucine and isoleucine biosynthesis pathways, respectively. TheilvG gene of K12 derivatives, including W3110 and ZGOLD5, contain apolar frameshift in the middle of the gene causing premature polypeptidechain termination (Parekh B S and Hatfield G W, J Bacteriol., 1997March; 179(6):2086-2088). This truncated protein is sensitive to valineand experiences feedback inhibition when valine is present. The pathwaysleading to the synthesis of isoleucine and valine are shut down in thepresence of valine even if the cell is starved for isoleucine. The mixedfeed, which includes yeast extract, compensates for the strain'sinability to produce isoleucine in the presence of valine. λ-indicatesthe absence of bacteriophage λ sequences in the lysogenic or lyticstate. ompT codes for a periplasmic endopeptidase that cleaves betweentwo consecutive base residues (White et al., J Biol Chem., 1995 Jun. 2;270(22):12990-4; and Dekker et al., Biochemistry. 2001 Feb. 13;40(6):1694-701). The ompT gene was removed from this strain to eliminateany potential degradation by OmpT (RES-10544). fhuA codes for amulti-functional outer membrane protein involved in the binding anduptake of ferrichrome and the attachment of T-odd phage (Ogata et al.,Uirusu. 2000 June; 50(1):17-26; and Moeck et al., J Bacteriol., 1995November; 177(21):6118-25). The gene was removed to prevent theinfection of the strain by T-odd phage, especially in fermentationtanks.

Expression vectors that are suitable for production of a desired proteinin prokaryotic cells typically comprise (1) prokaryotic DNA elementscoding for a bacterial origin for the maintenance of the expressionvector in a bacterial host; (2) DNA elements that control initiation oftranscription, such as a promoter; (3) DNA elements that control theprocessing of transcripts, such as a transcriptional terminator, and (4)a gene encoding a selectable marker, such as antibiotic resistance. Theprokaryotic host cell produces IL-29 upon introduction of an expressionvector and addition of an appropriate inducer. Accordingly, the presentinvention contemplates expression vectors comprising a promoter, theIL-29 optimized polynucleotide sequence, and a terminator sequence.Exemplary optimized IL-29 polynucleotide sequence are shown in SEQ IDNOs:1, 3, 5, 7, 9 and 11. In another embodiment, the expression vectorfurther comprises a selectable marker. In one embodiment, the selectablemarker is kanamycin resistance.

Expression vectors can also comprise polynucleotide sequences thatencode a peptide tag to aid in purification of the desired protein.Peptide tags that are useful for isolating recombinant polypeptidesinclude, for example, polyHistidine tags (which have an affinity fornickel-chelating resin), c-myc tags, calmodulin binding protein(isolated with calmodulin affinity chromatography), substance P, theRYIRS tag (which binds with anti-RYIRS antibodies), the Glu-Glu tag, andthe FLAG tag (which binds with anti-FLAG antibodies). See, for example,Luo et al., Arch. Biochem. Biophys. 329:215 (1996), Morganti et al.,Biotechnol. Appl. Biochem. 23:67 (1996), and Zheng et al., Gene 186:55(1997). Nucleic acid molecules encoding such peptide tags are available,for example, from Sigma-Aldrich Corporation (St. Louis, Mo.).

One of ordinary skill in the art will be familiar with a multitude ofmolecular techniques for the preparation of the expression vector. Forexample, the IL-29 polynucleotide can be prepared by synthesizingnucleic acid molecules using mutually priming, long oligonucleotides andthe nucleotide sequences described herein (see, for example, Ausubel etal., Short Protocols in Molecular Biology, 3^(rd) Edition, John Wiley &Sons, at pages 8-8 to 8-9 (1995)). Established techniques using thepolymerase chain reaction provide the ability to synthesize DNAmolecules at least two kilobases in length (Adang et al., Plant Molec.Biol. 21:1131 (1993), Bambot et al., PCR Methods and Applications 2:266(1993), Dillon et al., “Use of the Polymerase Chain Reaction for theRapid Construction of Synthetic Genes,” in Methods in Molecular Biology,Vol. 15: PCR Protocols: Current Methods and Applications, White (ed.),pages 263-268, (Humana Press, Inc. 1993), and Holowachuk et al., PCRMethods Appl. 4:299 (1995)).

Another method for constructing expression systems utilizes homologousrecombination using a yeast system. See U.S. Pat. No. 6,207,442, PlasmidConstruction by Homologous Recombination, incorporated herein byreference. The system provides a universal acceptor plasmid that can beused to clone a DNA encoding any polypeptide of interest, includingpolypeptide fusions. The system provides methods for preparing doublestranded, circular DNA molecules comprising a region encoding a proteinof interest. One or more donor DNA fragments encoding the protein ofinterest, i.e., IL-29, are combined with an acceptor plasmid, a firstDNA linker, and a second DNA linker in a Saccharomyces cerevisiae hostcell whereby the donor DNA fragment is joined to the acceptor plasmid byhomologous recombination of the donor DNA, acceptor plasmid, and linkersto form the closed, circular plasmid.

A nucleic acid molecule of the present invention can also be synthesizedwith “gene machines” using protocols such as the phosphoramidite method.If chemically-synthesized, double stranded DNA is required for anapplication such as the synthesis of a gene or a gene fragment, theneach complementary strand is made separately. The production of shortgenes (60 to 80 base pairs) is technically straightforward and can beaccomplished by synthesizing the complementary strands and thenannealing them. For the production of longer genes (>300 base pairs),however, special strategies may be required, because the couplingefficiency of each cycle during chemical DNA synthesis is seldom 100%.To overcome this problem, synthetic genes (double-stranded) areassembled in modular form from single-stranded fragments that are from20 to 100 nucleotides in length. For reviews on polynucleotidesynthesis, see, for example, Glick and Pasternak, MolecularBiotechnology, Principles and Applications of Recombinant DNA (ASM Press1994), Itakura et al., Annu. Rev. Biochem. 53:323 (1984), and Climie etal., Proc. Nat'l Acad. Sci. USA 87:633 (1990).

Examples of alternate techniques that can be used to prepare the IL-29gene and expression vector include, for example, restrictionendonuclease digestion and ligation, and polymerase chain reaction, allof which are well known in the art.

A wide variety of selectable marker genes is available (see, forexample, Kaufman, Meth. Enzymol. 185:487 (1990); Kaufman, Meth. Enzymol.185:537 (1990)). It is common for expression vectors to compriseselection markers, such as tetracycline resistance, ampicillinresistance, kanamycin resistance, neomycin resistance, orchloramphenicol resistance. A selectable marker will permit selectionand/or detection of cells that have been transformed with expressionvector from cells that have not been transformed. An expression vectorcan carry more than one such antibiotic resistance gene. An example ofselectable marker without antibiotic resistance uses the hok/sok systemfrom plasmid R1. The hok gene encodes the toxic Hok protein of 52 aminoacids and the sok gene encodes an antisense RNA, which is complementaryto the hok mRNA leader sequence. This selectable marker is known to oneskilled in the art and is described in more detail by Gerdes, K. et al.,Genetic Engineering, 19:49-61, 1997.

A wide variety of suitable recombinant host cells is encompassed by thepresent invention and includes, but is not limited to, gram-negativeprokaryotic host organisms. Suitable strains of E. coli include W3110,K12-derived strains MM294, TG-1, JM-107, BL21, and UT5600. Othersuitable strains include: BL21(DE3), BL21(DE3)pLysS, BL21(DE3)pLysE,DH1, DH4I, DH5, DH5I, DH5IF′, DH5IMCR, DH10B, DH10B/p3, DH11S, C600,HB101, JM101, JM105, JM109, JM110, K38, RR1, Y1088, Y1089, CSH18,ER1451, ER1647, E. coli K12, E. coli K12 RV308, E. coli K12 C600, E.coli HB101, E. coli K12 C600 R.sub.k-M.sub.k-, E. coli K12 RR1 (see, forexample, Brown (ed.), Molecular Biology Labfax (Academic Press 1991)).Other gram-negative prokaryotic hosts can include Serratia, Pseudomonas,Caulobacter. Prokaryotic hosts can include gram-positive organisms suchas Bacillus, for example, B. subtilis and B. thuringienesis, and B.thuringienesis var. israelensis, as well as Streptomyces, for example,S. lividans, S. ambofaciens, S. fradiae, and S. griseofuscus. Suitablestrains of Bacillus subtilus include BR151, YB886, MI119, MI120, andB170 (see, for example, Hardy, “Bacillus Cloning Methods,” in DNACloning: A Practical Approach, Glover (ed.) (IRL Press 1985)). Standardtechniques for propagating vectors in prokaryotic hosts are well-knownto those of skill in the art (see, for example, Ausubel et al. (eds.),Short Protocols in Molecular Biology, 3^(rd) Edition (John Wiley & Sons1995); Wu et al., Methods in Gene Biotechnology (CRC Press, Inc. 1997)).For an overview of protease deficient strains in prokaryotes, see,Meerman et al., Biotechnology 12:1107-1110, 1994.

Techniques for manipulating cloned DNA molecules and introducingexogenous DNA into a variety of host cells are disclosed by Sambrook etal., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989, and Ausubel et al.,eds., Current Protocols in Molecular Biology, John Wiley and Sons, Inc.,NY, 1987. Transformed or transfected host cells are cultured accordingto conventional procedures in a culture medium containing nutrients andother components required for the growth of the chosen host cells. Avariety of suitable media, including defined media and complex media,are known in the art and generally include a carbon source, a nitrogensource, essential amino acids, vitamins and minerals. Media may alsocontain such components as growth factors or serum, as required. Thegrowth medium will generally select for cells containing the exogenouslyadded DNA by, for example, drug selection or deficiency in an essentialnutrient that is complemented by the selectable marker carried on theexpression vector or co-transfected into the host cell. Liquid culturesare provided with sufficient aeration by conventional means, such asshaking of small flasks or sparging of fermentors. Transformed cells canbe selected and propagated to provide recombinant host cells thatexpress the gene of interest. IL-29 can be expressed in E. coli usingthe MBP (maltose binding protein) fusion system (New England Biolabs(NEB; Beverly, Mass.)). In this system, the IL-29 cDNA is attached tothe 3′ end of the malE gene to form an MBP-IL-29 fusion protein. Fusionprotein expression is driven by the tac promoter and is “off” until thepromoter is induced by addition of 1 mM IPTG (isopropylb-thiogalactosylpyranoside). The constructs can be built as in-framefusions with MBP in accordance with the Multiple Cloning Site (MCS) ofthe pMAL-c2 vector (NEB), and according to the manufacturer'sspecifications.

An E. coli expression vector has been constructed that contains a codonoptimized gene coding for human IL-29. This vector (pSDH175) vectorcontains the following functional elements: a lad repressor, tacpromoter, G10 translational enhancer, SmaI cloning site, transcriptionalterminator, Kanamycin selectable marker, and a pMB1 origin ofreplication and ROB gene. All of the plasmid numbers (vectors), asindicated in below Table 1 contain the same functional elements as thepSDH175 vector, but for the indicated substitutions of the translationalenhancer. The particular IL-29 molecule expressed by the variousplasmids or vectors of Table 1 is also indicated in the IL-29 constructcolumn. A number of changes to the protein structure of IL-29 were madeto enhance expression or improve refolding. The cysteine in position 172of the wild type IL-29 protein (SEQ ID NO:12) was changed to a serine(SEQ ID NO:2 or C172S) and the resulting expression vector was labeledpSDH177. The G10 translational enhancer of pSDH177 was replaced with theZymo2 vector (SEQ ID NO:13) and this vector was labeled pCHAN15. Theamino acids in position 2-7 of SEQ ID NO:2 were deleted from the pCHAN15vector to form the pTAP440 vector encoding this alternative form ofIL-29 as shown in SEQ ID NO:6. The polypeptide of SEQ ID NO:6 is alsoreferred herein as IL-29 C172S d2-7 or C172S d2-7. In vector pTAP438, aleucine was inserted into position 2, behind the N-terminal methionineof SEQ ID NO:2 and is shown as SEQ ID NO:4. The polypeptide of SEQ IDNO:4 is also referred to herein as IL-29 C172S Leu Insert or C172S LeuInsert. These vectors were transformed into a number of host strains toform the expression strains listed in Table 1 below. Recombinant humanIL-29 has been produced in fed batch fermentations using a number of E.coli production hosts and vectors. The IL-29 is produced as insoluble,retractile inclusion bodies in the different E. coli host strains used.

TABLE 1 Production Plasmid Translational Strain Host Strain Numberenhancer IL-29 construct EE669 W3110 pSDH175 G10 Codon optimizedwildtype (SEQ ID NO: 11) EE675 W3110 pSDH177 G10 Codon optimized C172S(SEQ ID NO: 1) EE698 W3110 pSDH 188 RBS2 Codon optimized ribosome- C172Sbinding site (SEQ ID NO: 1) EE826 W3110 pTAP440 Zymo2 C172S d2-7. Codonoptimized (SEQ ID NO: 5) EE708 ZGOLD1 pSDH188 RBS2 Codon optimizedribosome- C172S binding site (SEQ ID NO: 1) EE733 ZGOLD1 pCHAN15 Zymo2Codon optimized C172S (SEQ ID NO: 1) EE833 ZGOLD1 pTAP440 Zymo2 C172Sd2-7. Codon optimized (SEQ ID NO: 5) EE831 ZGOLD1 pTAP438 Zymo2 C172SLeu Insert (SEQ ID NO: 3) EE867 ZGOLD5 pTAP440 Zymo2 C172S d2-7. Codonoptimized (SEQ ID NO: 5) EE870 ZGOLD5 pCHAN15 Zymo2 Codon optimizedC172S (SEQ ID NO: 1)

High IL-29 expression levels have been obtained using E. coli ZGOLD5(EE867) cells, described below, containing the expression vectorpTAP440. A number of fed batch fermentation methods have been developedfor the production of IL-29 using this strain. Either a one stage or twostage seeding method can be used to start the fermentations. The seedmedium is a defined recipe (ZSM), described below in Table 2, containing2% glucose and is inoculated using vials from a working cell bank (WCB).The fermentation is started with an inoculum from an overnight (16-18hours) culture grown in ZSM. The production medium (PCOL18) is a definedsalts medium containing 1-2% glucose, 1% soy hydrolysate and 0.5% yeastextract. The initial batch phase is run for 7-8 hours, followed byglucose only feeding for the next 12 hours. The feed rate is maintainedconstant throughout the fermentation. The IL-29 expression is induced byaddition of isopropyl thiogalactopyranoside (IPTG) to a finalconcentration of 1 mM at 24 hours elapsed fermentation time (EFT). Totalfermentation time is about 48 hours.

TABLE 2 ZSM seed medium recipe per liter medium Amount Ingredient YeastExtract 5.0 g Sodium Sulfate dibasic 2.0 g Ammonium Sulfate dibasic 2.5g Ammonium Chloride 0.5 g Potassium Phosphate dibasic 14.6 g PotassiumPhosphate monobasic 3.6 g Deionized water QS to 1.0 L After autoclavingadd: 60% Glucose (wt/v) 33 mL Trace Elements Solution 3 mL 1M MgSO4 3 mLKanamycin 1.0 mL (25 mg/mL stock concentration)Fermentation

In one embodiment of the present invention batch fermentation can beused, particularly when large scale production of IL-29 using theexpression system of the present invention is required. Generally, batchfermentation comprises that a first stage seed flask is prepared bygrowing E. coli strains expressing IL-29 in a suitable medium in shakeflask culture to allow for growth to an optical density (OD) of 5 to 20at 600 nm. A suitable medium contains nitrogen from a source(s) such asammonium sulfate, ammonium phosphate, ammonium chloride, yeast extract,hydrolyzed animal proteins, hydrolyzed plant proteins or hydrolyzedcaseins. Phosphate will be supplied from potassium phosphate, ammoniumphosphate, phosphoric acid or sodium phosphate. Other components of themedium include magnesium chloride or magnesium sulfate, ferric sulfateor ferric chloride, and other trace elements. Growth medium can besupplemented with carbohydrates, such as fructose, glucose, galactose,lactose, and glycerol, to improve growth.

A first stage seed flask is prepared as follows: The IL-29 producing E.coli strains (e.g., W3110, ZGold1 and ZGold5) are grown in a suitablemedium in shake flask culture to allow for growth to an optical density(OD) of between 5 and 20 at 600 nm. A suitable medium can be, forexample: Super Broth II, APS-Super Broth, or ZSM. Growth medium can besupplemented with carbohydrates to improve growth. The preferredcarbohydrate additions can be, for example, glycerol or glucose addedfrom 1 to 20 g/L medium with a preference between 10-20 g/L. Growth isstarted by inoculating a shake flask (baffled flask from 500 ml to 3000ml) containing a preferred growth medium with E. coli containingkanamycin (10-50 ug/ml) from a frozen stock culture. Growth in the shakeflasks is at a temperature between 28 and 40° C. with a preference forgrowth between 30 and 37° C. The flasks are incubated with agitation setbetween 200 and 300 rpm.

A. Fed Batch Culture—Glucose Only Feeding (PCOL001 or PCOL0013)

Fermentation vessels are prepared with a suitable growth medium (forexample, see Table 3 below) and sterilized. The pH of the medium isadjusted to a pH between 6.2 and 7.2 with a preference for about pH 6.8.Growth medium can be supplemented with carbohydrates to improve growth.The preferred carbohydrate additions would be glycerol or glucose addedfrom 10 to 30 g/L medium with a preference between 10-20 g/L. Thevessels are set to the proper aeration and agitation levels andinoculated from a first stage seed flask culture or 2^(nd) stage seedvessel that has been grown between 10 and 20 hours and has an OD between5 and 20 at 600 nm. The inoculation level is between 1 and 5% (on avolume/volume basis) with a preference between 1 and 2% v/v. Thedissolved oxygen level is maintained above 20% saturation by increasingagitation speed, increasing the aeration rate, sparging in oxygen orvarious combinations thereof.

A carbohydrate solution is fed into the fermentor at a pre-determinedrate starting after 6-8 hours elapsed fermentation time (EFT). Thefeeding should be started no longer than 10 hours EFT. The feed iscontinued until the end of the fermentation. The feed solutions(glycerol or glucose) are prepared at 40-70% w/v with a preference for50% glucose (w/v). Feed rates can vary between 5-15 grams of glucose orglycerol per liter per hour, with a preference between 8-10 g/l/hr(starting volume). At a time between 20 and 30 hours EFT with apreference of 24 hours, IPTG is added to the culture to a concentrationof 1 mM. At a time between 48 and 56 hours EFT, the fermentation isharvested.

TABLE 3 PCOL13 Medium Recipe per Liter Medium Amount Ingredients priorto autoclavation Yeast extract 5.0 g (NH4)2SO4 9.9 g KH2PO4 1.75 gK2HPO4 12.25 g Antifoam AF204 0.1 mL Deionized water QS to 0.92 L PostSterilization additives: 1M MgSO4 10.0 mL Trace Elements Solution 34.0mL 60% Glucose (wt/vol) 33 mL 1M CaCl2 1 mL Kanamycin (25 mg/mL stock 1mL concentration)B. Fed Batch Culture—Glucose Only Feeding (PCOL0013+Additives)

Fermentation vessels are prepared with a suitable growth medium (forexample, see Table 3 above) and sterilized. The pH of the medium isadjusted to a pH between 6.2 and 7.2 with a preference for about pH 6.8.Growth medium can be supplemented with carbohydrates to improve growth.The preferred carbohydrate additions would be glycerol or glucose addedfrom 10 to 30 g/L medium with a preference between 10-20 g/L. Growthmedium can be supplemented with proteins to improve growth. Thepreferred protein additions are soy peptone and/or yeast extract addedfrom 5 to 30 g/L medium with a preference between 5-10 g/L. The vesselsare set to the proper aeration and agitation levels and inoculated froma first stage seed flask culture or 2^(nd) stage seed vessel that hasbeen grown between 10 and 20 hours and has an OD between 5 and 20 at 600nm. The inoculation level is between 1 and 5% (v/v) with a preferencebetween 1 and 2% v/v. The dissolved oxygen level is maintained above 20%saturation by increasing agitation speed, increasing the aeration rate,sparging in oxygen or various combinations.

A carbohydrate solution is fed into the fermentor at a pre-determinedrate starting after 6-8 hours elapsed fermentation time (EFT). Thefeeding needs to be started no longer than 10 hours EFT. The feed iscontinued until the end of the fermentation. The feed solutions(glycerol or glucose) are prepared at 40-70% w/v with a preference for50% glucose (w/v). Feed rates to can vary between 5-15 grams of glucoseor glycerol per liter per hour, with a preference between 8-10 g/l/hr(starting volume). At a time between 20 and 30 hours EFT with apreference of 24 hours, IPTG is added to the culture to a concentrationof 1 mM. At a time between 48 and 56 hours EFT, the fermentation isharvested.

C. Fed Batch Culture—Mixed Feed (PCOL18)

Fermentation vessels are prepared with a suitable growth medium andsterilized. The pH of the medium is adjusted to a pH between 6.2 and 7.2with a preference for about pH 6.8. Growth medium can be supplementedwith carbohydrates to improve growth. The preferred carbohydrateadditions would be glycerol or glucose added from 10 to 30 g/L mediumwith a preference between 10-20 g/L. The vessels are set to the properaeration and agitation levels and inoculated from a first stage seedflask culture or 2^(nd) stage seed vessel that has been grown between 10and 20 hours and has an OD between 5 and 20 at 600 nm. The inoculationlevel is between 1 and 5% (v/v) with a preference between 1 and 2% v/v.The dissolved oxygen level is maintained above 20% saturation byincreasing agitation speed, increasing the aeration rate, sparging inoxygen or various combinations.

A carbohydrate solution is fed into the fermentor at a pre-determinedrate starting after 6-8 hours elapsed fermentation time (EFT). Thefeeding needs to be started no longer than 10 hours EFT. The feedsolutions (glycerol or glucose) are prepared at 40-70% w/v with apreference for 50% glucose (w/v). Feed rates to be used can vary between5-15 grams of glucose or glycerol per liter per hour, with a preferencebetween 8-10 g/l/hr. At a time between 20 and 30 hours EFT with apreference of 24 hours, IPTG is added to the culture to a concentrationof 1 mM. At a time between 20 and 30 hours EFT with a preference of 24hours the glucose feed rate is decreased to 2-6 g/L/hr feed. A secondfeed containing yeast extract (20-30-% w/w) with a preference for 25%(w/w) is started at a rate between 2-6 g/L/hr with a preference for 2g/L/hr. At a time between 48 and 56 hours EFT, the fermentation isharvested.

IL-29 Recovery

Following fermentation the cells are harvested by centrifugation,re-suspended in deionized water and homogenized in an APV-Gaulinhomogenizer or other type of cell disruption equipment. Alternatively,the cells are taken directly from the fermentor, deionized water isadded, and then homogenized in an APV-Gaulin homogenizer. The homogenateis then centrifuged (either continuous or batch-mode), and the pelletcontaining the inclusion bodies is obtained after decanting thesupernatant. The inclusion body pellet is then washed in water, or Trisbuffers with or without varying levels of the following compounds:sodium chloride, urea, Triton X-100, sodium lauryl sulfate.

A. Homogenization and Pellet Washing (Direct Homogenization)

At the end of the fermentation run the temperature is adjusted downwardto between 4 and 20° C. The fermentation broth is harvested from thevessel and collection of the broth through the sample port.Alternatively, the broth can be pumped out through one of the sampleports. The fermentation broth can contain between 10-30% solids.

A homogenizer is used to disrupt the E. coli cells, but bead mills andsonicators can also be used. A homogenizer (APV-Gaulin 1000, APV 2000,or Niro Soavi) should be chilled to 4-15° C. prior to use. An equalamount of chilled deionized water is added to the fermentation broth.The fermentation broth is passed through the homogenizer and the cellsuspension is collected into a chilled container. The homogenizerpressure should be set between 8700-11,600 pounds per square inch(“psi”) (600-800 bar) for maximum cell disruption. The suspension ispassed through the homogenizer between 1-5 passes with a preference of 3passes.

B. Batch Harvest and Inclusion Body Washing

At the end of the homogenization process the disrupted cells aretransferred to 1 L centrifuge bottles, placing 0.75-1.0 L in each. ABeckman J6MI centrifuge with KompSpin KAJ7.100 rotor at 7,500 to16,000×G can be used to harvest the pellet. The Beckman Avanti JHCcentrifuge with the Beckman JLA-8.1 fixed angle rotor (7,500 to16,000×G) or the Aries JS 5.0 Swinging Bucket rotor with 2.25 L bottlesat 7,500 to 16,000×G can be used as well.

The bottles are centrifuged at 4° C. for 30 minutes. A centrifugationforce of 7500 to 16,000×G is used. The culture broth or supernatant ispoured off. Deionized water or buffer containing various additives isadded to the pellets. Additives can be Triton X 100 (0.1-5%), sodiumchloride (10-500 mM), zinc chloride (1-10 mM), EDTA (1-10 mM), sucrose(10-500 mM), sodium lauryl sulfate (0.1-2.0%) or urea (1-8 M). The washsolution is added in an equal volume to the supernatant decanted. Thepellets are re-suspended into the liquid by mixing with a spatulafollowed by mixing with a motorized mixing device such as the Omni EZhomogenizer. Mixing is performed until the IB pellet is well suspended.The solution is centrifuged at 7500-16,000×G, 4° C. for 30 minutes. Thebroth from the cell pellet is poured off and add water or buffer isadded to the pellets. After pellet re-suspension, the centrifugationstep is repeated and the supernatant poured off. This process can berepeated as many times as needed.

C. Continuous Cell Harvest and Inclusion Body Washing

At the end of the homogenization process the disrupted cells aretransferred to a chilled hold tank. The solution is passed through anappropriate continuous centrifuge such as a Carr or Westfalia disc stackcentrifuge. The solution can be passed through at feed rates between1-200 L per hour depending on the centrifuge used. The centrifugal forceof the centrifuge should be between 7,500 and 15,000×G. Fornon-discharging centrifuges such as a Carr Biopilot or Sharplesclarifier, the solution is passed through the centrifuge and the pelletscollected into the bowl. The inclusion body paste is scraped out of thebowl. The pellets can be used as is or re-diluted and passed through thecentrifuge again. The supernatant is discarded.

For continuous discharging centrifuges, such as a Westfalia C6 discstack centrifuge, the solution is passed through the centrifuge andsolids are kept in the bowl. The supernatant stream is continuouslydischarged. At predetermined set points, the solids in the bowl can bedischarged as a slurry into an appropriate collection vessel.Alternatively, water or buffer can be passed over the solids when theyare in the bowl to provide a washing step for the solids. The solids canthen be discharged at a pre-determined point.

Solubilization of Inclusion Bodies

The washed inclusion body pellet is solubilized in guanidinehydrochloride (4-6 M) containing dithiothreitol (DTT) at 10-50 mM.Solubilization is carried out for 1-2 hours at 15-25° C. The solubilizedmaterial is then clarified by centrifugation or used withoutclarification. HPLC analysis is performed to determine the amount ofIL-29 in the soluble fraction. Based on this concentration, theGuHCl/IL-29 solute will be diluted into a refold buffer mixture to afinal concentration between 1.25 and 2.0 mg/mL.

A. Solubilization of Washed Inclusion Bodies

The washed inclusion body prep can be solubilized using guanidinehydrochloride (5-8 M) or urea (7-8 M) containing a reducing agent suchas beta mercaptoethanol (10-100 mM) or dithiothreitol (5-50 mM). Thesolutions can be prepared in Tris, phosphate, HEPES or other appropriatebuffers. Inclusion bodies can also be solubilized in Tris buffer at pH10-11.5 with or without urea (1-2 M). Cells from 1 liter of fermentationbroth can be solubilized using 50-200 ml of the described solutions. Thepreferred method is to solubilize the washed inclusion body pellets from1 liter of fermentation broth in 150 ml of 6 M GuHCl prepared in 100 mMTris pH 8.0 containing 40 mM DTT. The slurry is re-suspended by mixingwith a spatula followed by homogenization with an Omni EZ homogenizer ormixing with a mechanical device. Incubate the mixture for 30-90 minuteswith mixing at 4-30° C. to finish the solubilization process. The samplecan be centrifuged at 7500-16,000×G at 4° C. for 10-30 minutes using anappropriate centrifuge. The supernatant sample is decanted and retained.Non-clarified samples can also be used for refolding.

B. Solubilization of Washed Inclusion Body Slurries

The washed inclusion body preparation can be produced as slurry ofinclusion bodies in water. This is typical after centrifugation andwashing using a continuous centrifuge. Solubilizing agents such asguanidine hydrochloride (4-6 M) or urea (4-7 M) can be added in drypowder form to the inclusion body slurries. Buffer (Tris, phosphate,HEPES), salts (magnesium chloride, sodium chloride, potassium chloride)and other compounds such as PEG 3500 can also be added in powder form tothe slurried mixture. Reducing agents such as beta mercaptoethanol(10-100 mM) or dithiothreitol (5-50 mM) can be added in powder or liquidform. The slurry is re-suspended by mixing with a high-powered mixer andimpeller, an Omni EZ homogenizer, or mixing with a mechanical device.The mixture is incubated for 30-90 minutes with mixing at 4-30° C. tofinish the solubilization process. The solubilized inclusion body slurrycan is then be centrifuged at 7500-16,000×G at 4° C. for 10-30 minutesusing an appropriate centrifuge. The supernatant sample is decanted andretained. Alternatively, the solution is used without clarification.

Refolding

The refolding is performed by slowly adding the solute solution to arefolding mixture of arginine, cystine, cysteine, DTT and salts. TheIL-29 solute can be added by batch or fed batch. The recombinant humanIL-29 refolding reaction is quenched by adjusting the pH to 5.8 to 6.1,preferably about 5.9. The acidified refold is diluted 4.25-fold in 25 mMsodium acetate, pH 5.6 to precipitate misfolded and unfolded proteinsand to condition the refold for loading to the capture column. Theprecipitate is allowed to settle overnight and then the supernatant isclarified through a depth filter train composed of a coarse (nominal 0.8μm) and fine (nominal 0.2 μm) filter in series.

The concentration of the IL-29 in the solubilized fraction is determinedby reversed phase HPLC. The determination of the refolding buffer volumeis based on the amount of solute and the concentration of IL-29 presentin the solute. The refolding buffer can contain a variety of salts andpolyethylene glycol (0.05-0.5%). Arginine (0.5 to 1.25 M) is used toprevent aggregation. The preferred level of arginine is 1.0 M with anIL-29 concentration of 2.0 mg/ml. An oxido shuffling system is used toinitiate disulfide bonding of the IL-29 molecule.

The oxido shuffling system is based on mixtures of reduced and oxidizedmolecules such as cysteine and cystine, DTT and cystine, reducedglutathione and oxidized glutathione, or DTT and oxidized glutathione.The ratio of reduced to oxidized glutathione or cystine can rangebetween 1:1 to 6:1 with a concentration range between 0.5 and 8 mM. Theoptimal concentration for refolding IL-29 is about 4 mM cysteine: 2 mMcystine.

The solute containing IL-29 is added rapidly (1-30 minutes), or slowly(0.5-5 hours) to the refolding buffer with mixing. The IL-29 can beadded in one addition, in multiple additions or fed in over time. TheIL-29 is added to the refolding mixture to a final concentration between0.5 mg/ml and 3.0 mg/ml, preferably 1.5 mg/ml and 2.0 mg/ml. Thetemperature range is between 4-30° C. The pH is between 7.3 and 8.5. Thevessel containing the refold mixture is left open to the atmosphere orcan be sparged with air or nitrogen during renaturation. The refoldingis allowed to take place for 1-26 hours after the end of the soluteaddition. Thereafter the refolding reaction is quenched by adjusting thesolution pH to 5.5-6.5, and preferably to pH 5.9.

Capture of Refolded IL-29

The clarified, diluted IL-29 is captured on a cation exchange column,e.g., ToyoPearl SP 550C (Tosoh Bioscience), at pH 5.5. The equilibrationbuffer is 50 mM sodium acetate, pH 5.5, and the bound IL-29 is elutedwith an increasing linear gradient to 2 M sodium chloride, in 50 mMsodium acetate, pH 5.5. The capture column eluate pool is adjusted to1.0 M (NH₄)₂SO₄ and then passed through a 0.45 μm filter to removeinsoluble material.

This step uses a cation exchange column to capture properly folded IL-29from a diluted and clarified refold solution. In order for IL-29 to bindto the column a dilution of the refolded solution is first carried out.Currently the refolded IL-29 is diluted 1.5- to 10-fold in water or lowionic strength buffer at pH 5-7. Preferably a 1:4.25 dilution is carriedout, using 25 mM sodium acetate, pH 5.6. The conductivity of thesolution after dilution should be not more than 30 mS/cm. A precipitateforms and is allowed to settle out of solution for 0.5-18 hr at 10-25°C. The settling preferably occurs for 10-16 hr at 16-22° C. The settledsupernatant is then filtered to remove any remaining precipitate insolution. A depth filter train, composed of a 0.8 μm nominal filter inseries with a 0.2 μm nominal filter, has been used to remove theprecipitate. One can also use a single depth filters or other filtertypes, such as a bag filter or a graded density filter, or combinationsof filters, to clarify the diluted refold supernatant. One can also usecentrifugation, either continuous or batch-mode, to remove theprecipitate.

Recombinant IL-29 in the refold solution is captured on a cationexchange column at pH 5.5. Typically the column contains ToyoPearl SP550C resin from Tosoh Bioscience. The column is equilibrated with 50 mMsodium acetate, pH 5.5, and then loaded with clarified, diluted refoldto a 1.0-17.5 g IL-29 per liter resin load factor. Preferably the columnis loaded with 5-15 g IL-29 per liter resin. After loading, the columnis washed with 2-5 CV of equilibration buffer to remove unboundmaterial, and bound IL-29 is then eluted with a linear 0-2M sodiumchloride gradient in 50 mM sodium acetate, pH 5.5. IL-29 elutes fromSP550C such that the eluate pool is at approximately 0.7-0.8 M sodiumchloride.

Many different cation exchange resins for this step, including othersulfopropyl resins such as SP Sepharose XL from GE Healthcare, or weakcation exchangers such as carboxymethyl, can be used as well asdifferent types of solid supports such as agarose or cellulose, anddifferent resin bead particle sizes. One could also run this column atdifferent pH's in the range from 5.0 to 7.0, and with different buffersand salts. Modified gradient or step elution strategies or formats maybe employed to elute recombinant human IL-29 from the column. One canalso use expanded bed chromatography to carry out this purification.

Purification of IL-29

The filtered and conditioned solution is loaded to a hydrophobicinteraction chromatography (“HIC”) column, e.g., ToyoPearl Super Butyl550C (Tosoh Bioscience), previously equilibrated with 50 mM sodiumacetate, 1.5 M (NH₄)₂SO₄, pH 5.5. The HIC column is washed withequilibration buffer to remove unbound material and then IL-29 is elutedwith a linear gradient to 50 mM sodium acetate, pH 5.5. The HIC eluatepool is diluted 6-fold in water then filtered and applied to a cationexchange column, SP Sepharose HP (GE Healthcare), equilibrated in 50 mMsodium acetate, 300 mM sodium chloride, pH 5.5. The high performancecation exchange column is washed with equilibration buffer and theneluted with a linear gradient to 50 mM sodium acetate, 800 mM sodiumchloride, pH 5.5. The eluate pool is then concentrated byultrafiltration in a tangential flow filtration system equipped with a 5kDa molecular weight cut-off polyether sulfone membrane. Theconcentrated product, IL-29 bulk intermediate, is filtered, aliquottedand stored at ≦−60° C.

A. Intermediate Purification of Recombinant Human IL-29

This step is designed to achieve further purification of IL-29, usinghydrophobic interaction chromatography (HIC) to remove host cellproteins and IL-29 hydrophobic variants. Typically ToyoPearl Super Butyl550C resin (Tosoh Bioscience) is used for this step. The resin isequilibrated with 50 mM sodium acetate, 1.5 M (NH₄)₂SO₄, pH 5.5. Thepool of IL-29 eluted from the capture column is adjusted to 1.0 M(NH₄)₂SO₄, via 2-fold dilution with 50 mM sodium acetate, 2.0 M(NH₄)₂SO₄, pH 5.5, and then passed through a 0.2 μm or 0.45 μm nominalfilter. The adjusted and filtered IL-29 is then loaded onto theequilibrated resin to a load factor between 1.0-20 g IL-29 per literresin, and preferably to ≦18 g IL-29 per liter resin. The column iswashed with equilibration buffer to remove unbound material and thenIL-29 is eluted with a linear gradient from 50 mM sodium acetate, 1.0 M(NH₄)₂SO₄ to 50 mM sodium acetate with no ammonium sulfate, at pH 5.5.IL-29 elutes from the column from approximately 0.75 M (NH₄)₂SO₄ to 0 M(NH₄)₂SO₄. All preceding steps are performed at 16-24° C.

One of ordinary skill in the art can use other hydrophobic interactionchromatography resins for this step, including but not limited to otherbutyl substituted resins, such as ToyoPearl Butyl 650M (TosohBioscience), or those substituted with phenyl, such as ToyoPearl Phenyl650M (Tosoh Bioscience) or Phenyl Sepharose Fast Flow (GE Healthcare).In addition, different types of solid supports, such as agarose orcellulose, and different resin bead particle sizes, may be used. One canalso run this column at different pH's, in the range from 5.0 to 9.0, atdifferent temperatures, and with different buffers and salts (sodiumsulfate, for example). Other concentrations of salt in the HIC Load (1.5M ammonium sulfate, for example) may be used to bind IL-29 to the HICresin by enhancing the hydrophobic effect, and other gradient or stepelution strategies may be employed to elute IL-29 from the column. Onecan also use displacement chromatography on HIC resins to purify IL-29,while leaving variants of increased hydrophobicity bound to the column.

B. Polish Purification of Recombinant Human IL-29

This step employs high performance cation exchange chromatography toremove charged variants from the IL-29 solution. The term“high-performance” refers to the ability to better resolve differentcharged components from one another due in large part to a reduced resinbead size. In the process described here, the high performance cationexchange resin bead is approximately 9-fold smaller than the cationexchange resin bead used for the lower resolution capture (SP550C) step.For this purification step, SP Sepharose HP resin (GE Healthcare) isused. The resin is equilibrated with 50 mM sodium acetate, 300 mM sodiumchloride, pH 5.5. The pool from the HIC column is diluted 6-fold inwater or low ionic strength buffer, then 0.2 μm filtered in preparationfor loading to the column. The adjusted and filtered IL-29 solution isthen loaded onto the equilibrated resin to a load factor between 1.0-50g IL-29 per liter resin, and preferably to between 15-30 g IL-29 perliter resin. The column is washed with equilibration buffer to removeunbound material and then IL-29 is eluted with a linear gradient to 50mM sodium acetate, 800 mM sodium chloride, pH 5.5. IL-29 elutes from thecolumn from approximately 0.4 M to 0.6 M sodium chloride.

One can use any of many different cation exchange resins for this step,including other sulfopropyl resins, or weak cation exchangers such ascarboxymethyl, as well as different types of solid supports such asagarose or cellulose, and different resin bead particle sizes. One canalso run this column at different pH's in the range from 5.0 to 7.0, andwith different buffers and salts. The addition of organic modifiers(such as 10% isopropanol or 10% ethanol) to the equilibration andelution buffers may be used to alter column resolution and selectivity.Modified gradient or step elution strategies may be employed to eluteIL-29 from the column.

C. Concentration of IL-29

This step is designed to concentrate the SP HP column eluate, generatingIL-29 bulk intermediate. The SPHP pool is concentrated approximately 2-4fold using a 5-kDa molecular weight cut-off polyether sulfone tangentialflow filtration (TFF) plate and frame membrane at a transmembranepressure of 15-25 psi. After the concentrated retentate is removed fromthe TFF system, the system is rinsed with buffer (such as 50 mM sodiumacetate, pH 5.5), and the rinse is combined with the concentratedretentate. This solution is then filtered through a 0.2 μm membrane,then filled in appropriate containers and stored in preparation for thesubsequent PEGylation reaction. One can use membranes of differentcomposition, such as those constructed of regenerated cellulose, and/orof different porosity, such as a 3 kDa molecular weight cut-off plateand frame membrane or a 10 kDa molecular weight cut-off hollow fibersystem, to accomplish this ultrafiltration step. Alternatively thisconcentration step may be skipped, if the SPHP pool is of sufficientIL-29 concentration to execute the PEGylation reaction described below.

D. Properties of Purified Recombinant Human IL-29 Bulk Intermediate

The purity of IL-29 bulk intermediate is at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% by sodium dodecyl sulfatepolyacrylamide gel analysis. Aggregates are less than 1.0%, less than0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%,less than 0.4, less than 0.3%, less than 0.2%, less than 0.1%, or lessthan 0.005% by size exclusion HPLC. Charge heterogeneity by cationexchange HPLC is about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% and thepurity measured by reversed phase HPLC is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99%.

The potency of IL-29 (same assays for PEG-IL-29) was measured using acell-based activity assay. The bioassay utilizes a 293 human embryonickidney (293 HEK) reporter cell line that was engineered to over-expressthe human IL-29 receptor, and contains a firefly luciferase reporterconstruct (KZ157), which includes ISRE and STAT binding elements placeddirectly upstream of the luciferase gene. The IL-29 receptor is aheterodimer consisting of IL-10 receptor β (IL-10Rβ) and IL-28 receptoralpha (IL-28Rα) subunits. Over-expression of the IL-29 receptor wasachieved by stable transfection of 293 HEK cells with the IL-28Rα cDNA,which along with endogenously expressed IL-10Rβ form the heterodimericIL-29 receptor. Binding of IL-29 (or PEG-IL-29) to the IL-29 receptoractivates the JAK/STAT signaling pathway and results in the formation ofthe intracellular transcription factor, ISGF3. Subsequent binding ofISGF3 to ISRE/STAT DNA sequence elements resulted in expression of thefirefly luciferase gene product. Recombinant human IL-29 was active inthe IL-29 cell-based potency bioassay.

In the bioassay, the assay cells were stimulated with IL-29 (orPEG-IL-29) for 4 hours and then lysed. After addition of a luciferasesubstrate luciferin to the lysed cells, luciferase expression wasmeasured indirectly in relative light units (RLU) using a luminometer. Acalibration curve was generated using a IL-29 (or PEG-IL-29) referencestandard, relating the luminescence signal to the concentration of theIL-29 reference standard, from which the potency of control and testsamples was calculated. Results were reported as relative potency unitsper milligram (RPU/mg) calculated relative to the reference standard. Adevelopment reference lot for IL-29 and PEG-IL-29 were assigned relativepotency units of one per milligram of protein (1 RPU/mg).

PEGylation of IL-29

IL-29 polypeptides, fusion, fragments, mutants, and variants of thepresent invention can be modified with polyethylene glycol (“PEG”), aprocess known as “PEGylation.” PEGylation of an IL-29 polypeptide can becarried out by any of the PEGylation reactions known in the art (see,for example, EP 0 154 316, Delgado et al., Critical Reviews inTherapeutic Drug Carrier Systems, 9:249 (1992), Duncan and Spreafico,Clin. Pharmacokinet. 27:290 (1994), and Francis et al., Int J Hematol68:1 (1998)). For instance, PEGylation can be performed by an acylationreaction or by an alkylation reaction with a reactive polyethyleneglycol molecule. In an alternative approach, IL-29 polypeptideconjugates are formed by condensing activated PEG, in which a terminalhydroxy or amino group of PEG has been replaced by an activated linker(see, for example, Karasiewicz et al., U.S. Pat. No. 5,382,657).

PEGylation by acylation typically requires reacting an active esterderivative of PEG with an IL-29 polypeptide. An example of an activatedPEG ester is PEG esterified to N-hydroxysuccinimide As used herein, theterm “acylation” includes the following types of linkages between IL-29polypeptide and a water-soluble polymer: amide, carbamate, urethane, andthe like. Methods for preparing PEGylated IL-29 by acylation willtypically comprise the steps of (a) reacting an IL-29 polypeptide withPEG (such as a reactive ester of an aldehyde derivative of PEG) underconditions whereby one or more PEG groups attach to IL-29, and (b)obtaining the reaction product(s). Generally, the optimal reactionconditions for acylation reactions will be determined based upon knownparameters and desired results. For example, the larger the ratio ofPEG:IL-29, the greater the percentage of polyPEGylated IL-29 product.

PEGylation by alkylation generally involves reacting a terminalaldehyde, e.g., propionaldehyde, butyraldehyde, acetaldehyde, and thelike, derivative of PEG with IL-29 polypeptide in the presence of areducing agent. PEG groups are preferably attached to the polypeptidevia a —CH2-NH2 group.

Derivatization via reductive alkylation to produce a monoPEGylatedproduct takes advantage of the differential reactivity of differenttypes of primary amino groups available for derivatization. Typically,the reaction is performed at a pH that allows one to take advantage ofthe pKa differences between the ε-amino groups of the lysine residuesand the α-amino group of the N-terminal residue of the protein. By suchselective derivatization, attachment of a water-soluble polymer thatcontains a reactive group such as an aldehyde, to a protein iscontrolled. The conjugation with the polymer occurs predominantly at theN-terminus of the protein without significant modification of otherreactive groups such as the lysine side chain amino groups.

Reductive alkylation to produce a substantially homogenous population ofmonopegylated IL-29 conjugate molecule can comprise the steps of: (a)reacting an IL-28 or IL-29 polypeptide with a reactive PEG underreductive alkylation conditions at a pH suitable to permit selectivemodification of the α-amino group at the amino terminus of the IL-29polypeptide, and (b) obtaining the reaction product(s). The reducingagent used for reductive alkylation should be stable in aqueous solutionand preferably be able to reduce only the Schiff base formed in theinitial process of reductive alkylation. Preferred reducing agentsinclude sodium borohydride, sodium cyanoborohydride, dimethylamineborane, trimethylamine borane, and pyridine borane.

For a substantially homogenous population of monopegylated IL-29conjugates, the reductive alkylation reaction conditions are those thatpermit the selective attachment of the water-soluble polymer moiety tothe N-terminus of IL-29 polypeptide. Such reaction conditions generallyprovide for pKa differences between the lysine amino groups and theα-amino group at the N-terminus. The pH also affects the ratio ofpolymer to protein to be used. In general, if the pH is lower, a largerexcess of polymer to protein will be desired because the less reactivethe N-terminal α-group, the more polymer is needed to achieve optimalconditions. If the pH is higher, the polymer: L-29 need not be as largebecause more reactive groups are available. Typically, the pH will fallwithin the range of 3-9, or 3-6. Another factor to consider is themolecular weight of the water-soluble polymer. Generally, the higher themolecular weight of the polymer, the fewer number of polymer moleculeswhich may be attached to the protein. For PEGylation reactions, thetypical molecular weight is about 2 kDa to about 100 kDa, about 5 kDa toabout 50 kDa, or about 12 kDa to about 40 kDa. The molar ratio ofwater-soluble polymer to IL-29 will generally be in the range of 1:1 to100:1. Typically, the molar ratio of water-soluble polymer to IL-29 willbe 1:1 to 20:1 for polyPEGylation, and 1:1 to 5:1 for monoPEGylation.

In preparation for a PEGylation reaction, the recombinant IL-29 bulkintermediate is thawed and transferred to a reaction vessel. Buffer fordilution, sodium cyanoborohydride reductant stock solution, andderivatized polyethylene glycol (“PEG”) (e.g., 20 kDa linearmethoxyPEG-propionaldehyde) stock solution, are added to the reaction tocreate a mixture with 5 g/L IL-29, 10 g/L derivatized PEG, and 20 mMsodium cyanoborohydride at pH 5.5 in 50 mM sodium acetate buffer. Thereaction is allowed to proceed with mixing for ˜18 hr at 16-20° C.

This step is used to covalently attach polyethylene glycol (PEG)molecules to IL-29, and preferentially a single PEG at the protein'samino-terminus. A PEG stock solution, composed of, for example, 20 kDalinear methoxyPEG-propionaldehyde (from, for example, Nippon Oil & Fat),at 20-200 mg/mL concentration in 50 mM sodium acetate, pH 4.5-6.5, andpreferably at 100 mg/mL concentration, pH 5.5, is prepared. A reductantstock solution, comprised of 5-500 mM sodium cyanoborohydride(preferably 100-200 mM) in 50 mM sodium acetate buffer, with pH in therange of 4.5-6.5 (preferably pH 5.5), is also prepared. The IL-29 bulkintermediate solution is transferred to a reaction vessel of appropriatesize. Buffer (such as 50 mM sodium acetate, pH 5.5), reductant stocksolution, and PEG stock solution are added sequentially in that ordersuch that the final mixture contains a 2-6 g/L IL-29 concentration, a1-to-4-fold molar excess of PEG over IL-29, and a 5-40 mM concentrationof sodium cyanoborohydride, at a pH ranging from 4.5-6.5. The reactionsolution is then mixed for 2-24 hr at 16-24° C. In a preferred case, thefinal reaction mixture contains IL-29 at 3-5 g/L concentration, with a2-fold molar excess of PEG relative to the IL-29 polypeptideconcentration, and with 10-20 mM sodium cyanoborohydride, at pH 5.5.This reaction is allowed to mix for 14-18 hr at 16-20° C.

One skilled in the art would know to use other PEG molecules, of longeror shorter chain length, to PEGylate the protein. For example, a 30 kDalinear methoxyPEG-propionaldehyde to PEGylate IL-29 under the conditionsdescribed above has also been used. One can use branched chain, ratherthan linear, PEG molecules in the reaction. The activated PEG may alsobe derivatized using other aldehydes, such as butyraldehyde, or may bederivatized with other amine-reactive compounds. Other site-specific PEGchemistries could be used to target other specific sites on IL-29 forderivatization. For the reactive aldehydes example, other reducingagents that would selectively reduce an imine to the amine may besubstituted for the sodium cyanoborohydride. Other reaction conditions,varying temperature, pH, salt composition and concentration, may also betried to enhance yields of the desired N-terminally monoPEGylatedspecies.

Purification of PEG-IL-29

Afterwards the pegylating IL-29, the reaction is diluted 2-fold with 50mM sodium acetate, pH 5.5 then 0.2 μm filtered and loaded to a secondcation exchange column (e.g, SP Sepharose HP (GE Healthcare)),equilibrated in 50 mM sodium acetate, 200 mM sodium chloride, pH 5.5.The high performance cation exchange column is washed with equilibrationbuffer and then eluted with a linear gradient to 50 mM sodium acetate,500 mM sodium chloride, pH 5.5. The eluate pool containing monoPEGylatedIL-29 is then concentrated by ultrafiltration in a tangential flowfiltration system equipped with a 5 kDa molecular weight cut-offpolyether sulfone membrane. After concentration, the retentate isdiafiltered against 7 diavolumes of formulation buffer to generatemonopegylated IL-29 (“PEG-IL-29”) bulk drug substance. The formulatedbulk drug substance is then 0.2 μm filtered, filled, and stored at ≦−60°C. for future use.

A. High Performance Cation Exchange Purification of PEG-IL-29

This step employs high performance cation exchange chromatography toseparate multi-PEGylated (containing two or more PEG groups per protein)and unPEGylated IL-29 proteins from the desired monoPEGylated species.Here, SP Sepharose HP resin (GE Healthcare) is used. The resin isequilibrated with 50 mM sodium acetate, 200 mM sodium chloride, pH 5.5.The reaction mixture is diluted 2-fold in water or low ionic strengthbuffer (preferably 50 mM sodium acetate, pH 5.5), then 0.2 μm filteredin preparation for loading to the column. The adjusted and filteredsolution is then loaded onto the equilibrated resin, which is thenwashed with equilibration buffer to remove unbound material. PEGylatedIL-29 proteins are eluted with a linear gradient to 50 mM sodiumacetate, 500 mM sodium chloride, pH 5.5. MonoPEGylated IL-29 elutes fromthe column from approximately 0.3 M to 0.5 M sodium chloride. A step to50 mM sodium acetate, 1 M sodium chloride, pH 5.5, is then used to eluteunPEGylated IL-29 from the column.

One of skill in the art would know to use any of many different cationexchange resins for this step, including other sulfopropyl resins, orweak cation exchangers such as carboxymethyl, as well as different typesof solid supports such as agarose or cellulose, and different resin beadparticle sizes. A skilled artisan would also know to run this column atdifferent pH's in the range from 5.0 to 7.0, and with different buffersand salts. Modified gradient or step elution strategies may be employedto elute PEGylated IL-29 from the column.

B. Concentration and Diafiltration of PEG-IL-29

This step is designed to concentrate the SP HP column eluate containingPEG-IL-29, and exchanging the solution into formulation buffer,generating PEG-IL-29 bulk drug substance. The SPHP pool is concentratedapproximately 10-15 fold using a 5 kDa molecular weight cut-offpolyether sulfone tangential flow filtration (TFF) plate and framemembrane. After concentration, the solution is diafiltered for 5-10diavolumes against formulation buffer. Both concentration and bufferexchange occur at transmembrane pressures in the range of 15-25 psi. Thebuffer exchanged concentrate is then removed from the TFF system, thesystem is rinsed with buffer, and the rinse is combined with theconcentrated retentate. This solution is then filtered through a 0.2 μmmembrane, then filled in appropriate containers and stored as bulk drugsubstance.

One can also use membranes of different composition, such as thoseconstructed of regenerated cellulose, and/or of different porosity, suchas a 3 kDa molecular weight cut-off plate and frame membrane or a 10 kDamolecular weight cut-off hollow fiber system, to accomplish thisultrafiltration/diafiltration step.

Additional Purification of IL-29 and PEG-IL-29

It may be necessary to further purify either IL-29 or PEG-IL-29 toremove remaining impurities and contaminants. An anion exchange columnmay be used to reduce the endotoxin level. IL-29 is diluted to aconductivity level of <10 mS/cm and the pH is adjusted to 8.0. It isapplied to a Q Sepharose FF column that has been equilibrated to 20 mMTris, pH 8.0. The IL-29 should pass through the column and have anapproximately 80% reduction in endotoxin compared to the load. IL-29polypeptide or PEG-IL29 polypeptide will have an endotoxin level of lessthan 10 endotoxin units per milligram of IL-29 polypeptide or PEG-IL29polypeptide in a Limulus amoebocyte lysate assay based on USP <85> (See,for example, R. Nachum and R. N. Berzofsky, J. Clinical Microbiology,21(5):759-763 (May 1985)). Mustang Q or Mustang E charged membranes(Pall) may also be used to reduce endotoxin levels in solutions betweenpH 5.0 and 9.0.

Other purification steps that can be used to further purify IL-29include immobilized metal affinity chromatography, anion exchangechromatography, or hydrophobic charge induction chromatography. One maybe able to use displacement chromatography to purify IL-29 or PEG-IL-29,whereby high protein loading of the column causes the protein ofinterest to elute due to being displaced by more tightly bindingimpurities. Alternatively, it may be possible to utilize a flow-throughmode of chromatography whereby impurities bind to the resin, while IL-29or PEG-IL-29 pass through during the load step (as in the endotoxinremoval example on Q Sepharose, above).

Properties of Purified PEG-IL-29

The purity of PEG-IL-29 bulk drug substance is at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or greater than 99% bysodium dodecyl sulfate polyacrylamide gel analysis. Aggregates are lessthan 1.0%, less than 0.9%, less than 0.8%, less than 0.7%, less than0.6%, less than 0.5%, less than 0.4, less than 0.3%, less than 0.2%,less than 0.1%, or less than 0.005% by size exclusion HPLC. Chargeheterogeneity by cation exchange HPLC is about 10%, 9%, 8%, 7%, 6%, 5%,4%, 3%, 2% or 1% and the monoPEG purity measured by reversed phase HPLCis at least 95%, at least 96%, at least 97%, at least 98%, at least 99%,or greater than 99%. The potency of PEG-IL-29 was measured using acell-based activity assay as described above. PEG-IL-29 was active inthe IL-29 cell-based potency bioassay.

The invention is further illustrated by the following non-limitingexamples.

EXAMPLES Example 1 Construction of Expression Vector, pTAP395

The backbone used to construct the E. coli expression vector for IL-29C172S d2-7 (SEQ ID NO:5) was pTAP395. pTAP395 contained the srppromoter, two transcriptional terminators, rrnB T1 and rrnB T2,kanamycin resistance gene, origin of replication, URA3 selection markerand the ARS-CENS6 locus for plasmid replication in yeast. pTAP395 wasgenerated from pTAP238 but has a different translational enhancer frompTAP238. pTAP395 had the translational enhancer known as chant or zymo2(SEQ ID NO:13). pTAP395 was constructed using oligos zc42188 (SEQ IDNO:14), zc42187 (SEQ ID NO:15), zc42194 (SEQ ID NO:16), and zc29741 (SEQID NO:17) by implementing an overlap-PCR strategy. The ends of the PCRfragment were homologous to pTAP395. The central region between the XbaIand SmaI sites contained the zymo2 ((SEQ ID NO:13)) translationalenhancer. The PCR reagent concentrations were as follows: 1 μM ofzc42188 (SEQ ID NO:14) and zc29741 (SEQ ID NO:17); 50 nM of zc42187 (SEQID NO:15) and zc42194 (SEQ ID NO:16); 0.2 mM dNTPs; 1× reaction buffer;and 0.05 U/μL Pwo (Roche). The reaction consisted of 10 cycles of thefollowing: 94° C. for 30 seconds, 55° C. for 30 seconds, and 72° C. for30 seconds. Four reactions were done in all. The DNA was precipitatedusing two volumes of 100% ethanol and centrifuging in amicro-centrifuge. The supernatant was discarded, and the pellet wasresuspended in 10 μL of water. The resulting DNA fragment was checkedfor size by electrophoresis of 2 μL on a 2% 1×TBE agarose gel. The sizeof the PCR fragment was approximately 150 bp, as expected. The remainingDNA was mixed with 100 ng of pTAP238 digested with SmaI. The DNA mixturewas then mixed with 100 μL of electrocompetent SF838-9Dα yeast cells (S.cerevisiae) and electroporated under the following conditions: 25 μF,0.75 kV, and ∞ ohms. Six hundred microliters of 1.2 M sorbitol wereadded to the cells, which were then spread on −Ura D plates andincubated at 30° C. for approximately 72 hours. The Ura+ yeasttransformants from a single plate were suspended in 2-3 mL H₂O andcentrifuged briefly to pellet the cells. The cell pellet was resuspendedin 1 mL of lysis buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mMTris, pH 8.0, 1 mM EDTA). Five hundred microliters of the lysis mixturewere added to an Eppendorf tube containing 300 μL acid washed glassbeads and 500 μL phenol-chloroform. The sample was vortexed for 1-minuteintervals two or three times and then centrifuged for 5 minutes in anEppendorf centrifuge at maximum speed. Three hundred microliters of theaqueous phase were transferred to a fresh tube. The DNA was precipitatedwith 600 μL 100% ethanol and centrifuged for 10 minutes at 4° C. The DNApellet was resuspended in 100 μL H₂O. Forty microliters ofelectrocompetent MC1061 cells were transformed with 1 μL of the plasmidDNA prep. The cells were pulsed at 2.0 kV, 25 μF and 400 ohms. Followingelectroporation, 600 μL SOC were added to the cells which were allowedto recover at 37° C. for one hour. The cells were then plated as onealiquot onto LB plates (LB broth (Lennox), 1.8% Bacto™ Agar (Difco))containing 25 μg/mL kanamycin and grown overnight at 37° C. Screeningvia colony PCR using primers, zc42188 (SEQ ID NO:14) and zc29741 (SEQ IDNO:17), identified cells harboring the correct construct containing theDNA sequence for the altered translational enhancer. The PCR conditionswere as follows: 0.2 μM of each oligo; 0.2 mM dNTPs; 1× reaction buffer;and 0.05 U/μL Taq (Roche). The template for each reaction was a singlecolony picked from the transformation plate and suspended in 10 μL ofLB. The PCR consisted of 25 cycles of the following: 94° C. for 30seconds, 55° C. for 30 seconds, and 72° C. for 1 minute. All eightclones were positive as judged by analysis on a 2% agarose gel, andthree were subjected to sequence analysis. The correct clone becameknown as pTAP395.

Example 2 Construction of Codon Optimized IL-29 Gene

The IL-29 coding sequence with codon optimized for translation in E.coli was constructed from ten overlapping oligonucleotides (Oligonumber: zc44,559 (SEQ ID NO:18), zc44,566 (SEQ ID NO:19), zc44,565 (SEQID NO:20), zc44,562 (SEQ ID NO:21), zc44,563 (SEQ ID NO:22), zc44,560(SEQ ID NO:23), zc44,561 (SEQ ID NO:24), zc44,564 (SEQ ID NO:25),zc44,557 (SEQ ID NO:26) and zc44,558 (SEQ ID NO:27). Primer extensionfollowed by PCR amplification produced a full-length, optimized IL-29gene. The final PCR product was inserted into the cloning vector,pCR-Blunt II TOPO by ligation. The ligation mix was transformed intocompetent E. coli TOP10. Kanamycin resistant clones were screened bycolony PCR. A positive clone was verified by DNA sequencing.

Example 3 Construction of Expression Vector pCHAN15

The strategy used to generate the IL-29 C172S (SEQ ID NO:1) mutant isbased on the QuikChange® Site-Directed Mutagenesis Kit (Stratagene, LaJolla, Calif.). Primers were designed to introduce the C172S mutationaccording to the manufacturer's suggestions. The primers were designatedzc44,340 (SEQ ID NO:28) and zc44,341 (SEQ ID NO:29). PCR was performedto generate the IL-29 C172S mutant according to instructions providedwith the QuikChange Mutagenesis Kit. Five identical 50 μL reactions wereset-up. Each reaction contained 2.5 μL of pSDH175 (expression constructwith the optimized IL-29 gene sequence) as template. The PCR cocktailcontained the reagents: 30 μL 10×PCR buffer, 125 ng (27.42 μL) zc44,340(SEQ ID NO:28). 125 ng (9.18 μL) zc44,341 (SEQ ID NO:29), 6 μL dNTP, 6μL Pfu Turbo polymerase (Strategene), and 206.4 μL water. Each reactionreceived 47.5 μL of the cocktail. The PCR conditions were as follows: 1cycle of 95° C. for 30 seconds followed by 16 cycles of 95° C. for 30seconds, 55° C. for 1 minute, 68° C. for 7 minutes, and 1 cycle at 68°C. for 7 minutes. After the last cycle, the reaction was held at 4° C.All five PCR reactions were consolidated into one tube. As permanufacturer's instructions, 5 μL of the restriction enzyme DpnI wasadded to the PCR reaction and the mixture was incubated at 37° C. for 2hours. DNA was precipitated by adding 10% 3M sodium acetate and twovolumes of 100% ethanol. The DNA pellet was resuspended in 20 μL waterand transformed into E. coli strain DH10B. Electroporated cells werethen allow to recover at 37° C. for 1 hour. The cells were plated on anLB agar containing 25 μg/mL kanamycin and incubated at 37° C. overnight.Ten clones were screened for the presence of an insert containing IL-29C172S. DNA was isolated from all ten clones using the QIAprep™ SpinMiniprep Kit (Qiagen) and analyzed for presence of insert by digestionwith XbaI (Roche) and PstI (New England Biolabs). Nine clones containedthe insert and were sequenced to insure that the IL-29 C172S mutationhad been introduced. One clone for which the sequence was verified wassaved and labeled pSDH188. Subsequently, the IL-29 C172S insert wassub-cloned into the expression vector, pTAP395. The resulting constructbecame known as pCHAN15.

Example 4 Construction of Expression Vector, pTAP440

The oligos used for the construction of pTAP440 were zc49249 (forwardprimer) (SEQ ID NO:30) and zc45403 (reverse primer) (SEQ ID NO:31). Thefirst 38 bases on the 5′ end of zc49249 are homologous to the vectorbackbone, pTAP395. The remaining 26 bases contained the initialmethionine codon (ATG) followed by DNA sequence homologous to the IL-29gene, starting at the eighth codon. The 5′ end of the reverse primer,zc45403 (SEQ ID NO:31), consists of 39 bases homologous to the vectorbackbone. The second half, 25 bases, of the oligo are homologous to theoptimized IL-29 gene which contained the base pair changes coding forthe C172S mutation. To amplify the gene for IL-29 the following finalconcentrations of reagents were used in a total reaction volume of 100μL: 0.2 μM of each oligo; 0.2 mM dNTPs; 1× reaction buffer; 10% DMSO;and 0.05 U/μL Pwo (Roche). The template used for amplification waspCHAN15. The reaction consisted of 25 cycles of the following: 94° C.for 30 seconds, 55° C. for 30 seconds, and 72° C. for 1 minute. The DNAwas precipitated using two volumes of 100% ethanol and pelleted in amicro-centrifuge. The supernatant was discarded, and the pellet wasresuspended in 10 μL of water. The resulting DNA fragment was checkedfor size by electrophoresis of 2 μL on a 1% 1×TBE agarose gel. The sizeof the PCR fragment was approximately 500 bp, as expected. Eightmicroliters of the DNA were mixed with 2 μL of pTAP395 digested withSmaI.

The DNA mixture was mixed with 100 μL of electrocompetent SF838-9Dαyeast cells (S. cerevisiae) and electroporation was performed under thefollowing conditions: 25 μF, 0.75 kV, and ∞ ohms. Six hundredmicroliters of 1.2 M sorbitol were added to the cells. The cells werespread on −Ura D plates and incubated at 30° C. for approximately 72hours. The Ura+transformants from a single plate were resuspended in 2-3mL H₂O and centrifuged briefly to pellet the cells. The cell pellet wasresuspended in 1 mL of lysis buffer (2% Triton X-100, 1% SDS, 100 mMNaCl, 10 mM Tris, pH 8.0, 1 mM EDTA). Five hundred microliters of thelysis mixture were added to an Eppendorf tube containing 300 μL acidwashed glass beads and 500 μL phenol-chloroform. The sample was vortexedfor 1-minute intervals two or three times and centrifuged for 5 minutesin an Eppendorf centrifuge at maximum speed. Three hundred microlitersof the aqueous phase were transferred to a fresh tube. The DNA wasprecipitated with 600 μL of 100% ethanol and centrifuged for 10 minutesat 4° C. The DNA pellet was resuspended in 50 μL H₂O.

Transformation of electrocompetent E. coli cells was performed using 1μL of the plasmid DNA prep and 40 μL of MC1061 cells. The cells werepulsed at 2.0 kV, 25 μF and 400 ohms. Following electroporation, 600 μLof Terrific Broth were added to the cells which were allowed to recoverat 37° C. for one hour. The cells were plated on LB agar containing 25μg/mL kanamycin and grown overnight at 37° C. Screening via colony PCRusing primers, zc49249 (SEQ ID NO:30) and zc45403 (SEQ ID NO:31),identified the cells harboring the correct expression construct whichcontained the DNA sequence for IL-29 C172S d2-7 (SEQ ID NO:5). The PCRconditions were as follows: 0.2 μM of each oligo; 0.2 mM dNTPs; 1×reaction buffer; 10% DMSO; and 0.05 U/μL Pwo (Roche).

The template for each reaction was a single colony picked from thetransformation plate and suspended in 10 μL of LB. The PCR consisted of25 cycles of the following: 94° C. for 30 seconds, 55° C. for 30seconds, and 72° C. for 1 minute. All eight clones tested were positiveby analysis on a 1% agarose gel, and four were subjected to sequenceanalysis. One of the clones, now known as pTAP440, was selected from thefour submitted to sequencing. Ten microliters of DNA were digested in areaction that contained 2 μL of NotI, 3 μL of NEB buffer 3, and 15 μL ofwater for one hour at 37° C. Then 7 μL of this reaction were mixed with2 μL of 5× buffer and 1 μL of T4 DNA ligase. This reaction incubated atroom temperature for one hour. One microliter of the ligation reactionwas used to transform strain W3110 [F⁻ IN(rrnD⁻ rrnE)1 lambda⁻](obtained, for instance, from ATCC) by electroporation.

The cells were pulsed at 2.0 kV, 25 μF and 400 ohms. Followingelectroporation, 600 μL SOC (2% Bacto™ Tryptone (Difco, Detroit, Mich.),0.5% yeast extract (Difco), 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl₂, 10 mMMgSO₄, 20 mM glucose) were added to the cells. The cells grew at 37° C.for one hour and were plated in one aliquot on an LB agar containing 25μg/mL kanamycin. The plate was incubated at room temperature for 48hours. Four colonies were picked and grown overnight in LB containing 25μg/mL kanamycin at 37° C. Plasmid DNA was isolated using a QIAprep SpinMiniprep Kit (Qiagen). The DNA was digested with PvuII to confirm theloss of yeast URA3 and CEN/ARS elements: 12.5 μL DNA, 1 μL PvuII, 1.5 μLbuffer 2 NEB at 37° C. for one hour. One of the correct clones lackingthe yeast elements was used to transform 40 μL electrocompetent ZGOLD5[F− IN(rrnD-rrnE)1 lambda⁻ ΔompT::tet ΔfhuA::Cm]. The cells were pulsedat 2.0 kV, 25 μF and 400 ohms. Following the electroporation, 600 μL SOCwere added to the cells. The cells were grown at 37° C. for one hour andthen plated on LB agar containing 25 μg/mL kanamycin. The plates wereincubated at 37° C. for 24 hours. Since the bacteria were transformedwith pure plasmid, it was assumed that kanamycin resistant bacteriaharbored the plasmid. ZGOLD5 transformed with pTAP440 was preserved andstored frozen.

Example 5 Glucose Fed Batch Fermentation ECD686 (IL-29: C172S)

A shake flask (baffled 500 ml flask with 100 ml medium) was preparedwith ZSM medium. Growth was started by inoculating the shake flask witha 0.10 mL of E. coli W3110 containing the expression vector pSDH177(EE675) from a vial of a research working cell bank. This vector codesfor the C172S form of the IL-29 molecule. Growth in the shake flask wasat a temperature of 32° C. with the agitation set at 250 rpm. Theculture was grown overnight (16 hours) until the OD₆₀₀ was between Band20 units.

A 6 L fermentation vessel was prepared with 3.0 L of PCOL-01 medium andsterilized. After sterilization the medium was supplemented with glucoseat 20 g/L, 1 M MgSO4 (10 ml/L) and kanamycin at 25 ug/ml. The pH of themedium was adjusted to a pH of 6.8. The vessel aeration was set to 1 vvmand agitation at 350 rpm, temperature was set to 37° C. The fermentorwas inoculated from a shake flask culture that had been grown for 16hours and had an OD of 10.9 at 600 nm. The inoculation level was 5%volume/volume. The dissolved oxygen level was maintained above 30%saturation by increasing agitation speed.

A carbohydrate solution was fed into the fermentor starting at 9 hoursEFT. The feed was continued until the end of the fermentation. The feedsolution was glucose prepared at 50% w/w. The feed rate was 10.8 gramsof glucose per liter per hour based on the initial starting volume. At24 hours EFT, IPTG was added to the culture to a concentration of 1 mM.At 48 hours EFT, the fermentation was harvested. The biomass reached58.8 g dry cell weight (DCW)/L at harvest with a fermentation titer of4.6 g IL-29/L fermentor broth.

Example 6 Glucose Fed Batch Fermentation ECD712 (IL-29 C172S)

A shake flask (baffled 500 ml flask with 100 ml medium) was preparedwith ZSM medium. Growth was started by inoculating the shake flask witha 0.10 mL of E. coli ZGOLD1 [F− IN(rrnD-rrnE)1 lambda⁻ ΔompT::tet]containing the expression vector pCHAN15 (EE733) from a vial of aresearch working cell bank. This vector codes for the C172S form of theIL-29 molecule. Growth in the shake flask was at a temperature of 32° C.with the agitation set at 250 rpm. The culture was grown overnight (16hours) until the OD₆₀₀ was between 8 and 20 units.

A 6 L fermentation vessel was prepared with 3.0 L of PCOL-01 medium andsterilized. After sterilization the medium was supplemented with glucoseat 20 g/L, 1 M MgSO4 (10 ml/L) and kanamycin at 25 ug/ml. The pH of themedium was adjusted to a pH of 6.8. The vessel aeration was set to 1 vvmand agitation at 350 rpm, temperature was set to 37° C. The fermentorwas inoculated from a shake flask culture that has been grown for 16hours and had an OD of 13.1 at 600 nm. The inoculation level was 5% v/v.The dissolved oxygen level was maintained above 30% saturation byincreasing agitation speed.

A carbohydrate solution was fed into the fermentor starting at 9.5 hoursEFT. The feed was continued until the end of the fermentation. The feedsolution was glucose prepared at 50% w/w %. The feed rate was 9.5 gramsof glucose per liter per hour based on the initial starting volume. At24 hours EFT, IPTG was added to the culture to a concentration of 1 mM.At 48 hours EFT, the fermentation was harvested. The biomass reached67.4 g dry cell weight (DCW)/L at harvest with a fermentation titer of3.6 g IL-29/L fermentor broth.

Example 7 Glucose Fed Batch Fermentation ECD856 (IL-29: C172S+Leucine)

A shake flask (baffled 500 ml flask with 100 ml medium) was preparedwith ZSM medium. Growth was started by inoculating the shake flask witha 0.10 mL of E. coli ZGOLD1 containing the expression vector pTAP438(EE831) from a vial of a research working cell bank. This vector codesfor a C172S form of the IL-29 molecule that contains an added leucineafter the N-terminal methionine. Growth in the shake flask was at atemperature of 32° C. with the agitation set at 250 rpm. The culture wasgrown overnight (16 hours) until the OD₆₀₀ was between Band 20 units.

A 6 L fermentation vessel was prepared with 3.0 L of PCOL-13 medium andsterilized. After sterilization the medium was supplemented with glucoseat 20 g/L, 1 M MgSO4 (10 ml/L) and kanamycin at 25 ug/ml. The pH of themedium was adjusted to a pH of 6.8. The vessel aeration was set to 1 vvmand agitation at 350 rpm, temperature was set to 37° C. The fermentorwas inoculated from a shake flask culture that has been grown for 16hours and had an OD of 13.8 at 600 nm. The inoculation level was 5%volume/volume. The dissolved oxygen level was maintained above 30%saturation by increasing agitation speed.

A carbohydrate solution was fed into the fermentor starting at 8 hoursEFT. The feed was continued until the end of the fermentation. The feedsolution was glucose prepared at 50% w/w %. The feed rate was 9.5 gramsof glucose per liter per hour based on the initial starting volume. At24 hours EFT, IPTG was added to the culture to a concentration of 1 mM.At 50 hours EFT, the fermentation was harvested. The biomass reached70.0 g DCW/L at harvest with a fermentation titer of 7.3 g IL-29/Lfermentor broth.

Example 8 Glucose Fed Batch Fermentation ECD859 (IL-29 C172S d2-7)

A shake flask (baffled 500 ml flask with 100 ml medium) was preparedwith ZSM medium. Growth was started by inoculating the shake flask witha 0.10 mL of E. coli ZGOLD1 containing the expression vector pTAP440(EE833) from a vial of a research working cell bank. This vector codesfor a form of the IL-29 molecule that contains a deletion of the secondthrough seventh amino acids. Growth in the shake flask was at atemperature of 32° C. with the agitation set at 250 rpm. The culture wasgrown overnight (16 hours) until the OD₆₀₀ was between 8 and 20 units.

A 6 L fermentation vessel was prepared with 3.0 L of PCOL-13 medium andsterilized. After sterilization the medium was supplemented with glucoseat 20 g/L, 1 M MgSO4 (10 ml/L) and kanamycin at 25 ug/ml. The pH of themedium was adjusted to a pH of 6.8. The vessel aeration was set to 1 vvmand agitation at 350 rpm, temperature was set to 37° C. The fermentorwas inoculated from a shake flask culture that has been grown for 16hours and had an OD of 12.5 at 600 nm. The inoculation level was 5% v/v.The dissolved oxygen level was maintained above 30% saturation byincreasing agitation speed.

A carbohydrate solution was fed into the fermentor starting at 8 hoursEFT. The feed was continued until the end of the fermentation. The feedsolution was glucose prepared at 50% w/w. The feed rate was 9.5 grams ofglucose per liter per hour based on the initial starting volume. At 24hours EFT, IPTG was added to the culture to a concentration of 1 mM. At50 hours EFT, the fermentation was harvested. The biomass reached 82.8 gdry cell weight (DCW)/L at harvest with a fermentation titer of 11.3 gIL-29/L fermentor broth.

Example 9 Glucose Fed Batch Fermentation+Soy Peptone ECD892 (IL-29 C172Sd2-7)

A shake flask (baffled 500 ml flask with 100 ml medium) was preparedwith ZSM medium. Growth was started by inoculating the shake flask witha 0.10 mL of E. coli W3110 containing the expression vector pTAP440(EE826) from a vial of a research working cell bank. This vector codesfor a form of the IL-29 molecule that contains a deletion of the secondthrough seventh amino acids. Growth in the shake flask was at atemperature of 32° C. with the agitation set at 250 rpm. The culture wasgrown overnight (16 hours) until the OD₆₀₀ was between Band 20 units.

A 6 L fermentation vessel was prepared with 3.0 L of PCOL-13 medium,containing 10.0 g/L of soy hydrolysate, and sterilized. Aftersterilization the medium was supplemented with glucose at 20 g/L, 1 MMgSO4 (10 ml/L) and kanamycin at 25 ug/ml. The pH of the medium wasadjusted to a pH of 6.8. The vessel aeration was set to 1 vvm andagitation at 350 rpm, temperature was set to 37° C. The fermentor wasinoculated from a shake flask culture that has been grown for 16 hoursand had an OD of 11.5 at 600 nm. The inoculation level was 5%volume/volume. The dissolved oxygen level was maintained above 30%saturation by increasing agitation speed.

A carbohydrate solution was fed into the fermentor starting at 8 hoursEFT. The feed was continued until the end of the fermentation. The feedsolution was glucose prepared at 50% w/w %. The feed rate was 9.5 gramsof glucose per liter per hour based on the initial starting volume. At24 hours EFT, IPTG was added to the culture to a concentration of 1 mM.At 50 hours EFT, the fermentation was harvested. The biomass reached73.3 g dry cell weight (DCW)/L at harvest with a fermentation titer of12.5 g IL-29/L fermentor broth.

Example 10 Glucose Fed Batch Fermentation+Yeast Extract ECD880 (IL-29C172S d2-7)

A shake flask (baffled 500 ml flask with 100 ml medium) was preparedwith ZSM medium. Growth was started by inoculating the shake flask witha 0.10 mL of E. coli ZGOLD1 containing the expression vector pTAP440(EE833) from a vial of a research working cell bank. This vector codesfor a form of the IL-29 molecule that contains a deletion of the secondthrough seventh amino acids. Growth in the shake flask was at atemperature of 32° C. with the agitation set at 250 rpm. The culture wasgrown overnight (16 hours) until the OD₆₀₀ was between Band 20 units.

A 6 L fermentation vessel was prepared with 3.0 L of PCOL-13 medium,containing 20.0 g/L of yeast extract, and sterilized. Aftersterilization the medium was supplemented with glucose at 20 g/L, 1 MMgSO₄ (10 ml/L) and kanamycin at 25 ug/ml. The pH of the medium wasadjusted to a pH of 6.8. The vessel aeration was set to 1 vvm andagitation at 350 rpm, temperature was set to 37° C. The fermentor wasinoculated from a shake flask culture that has been grown for 16 hoursand had an OD of 8.9 at 600 nm. The inoculation level was 1%volume/volume. The dissolved oxygen level was maintained above 30%saturation by increasing agitation speed.

A carbohydrate solution was fed into the fermentor starting at 8 hoursEFT. The feed was continued until the end of the fermentation. The feedsolution was glucose prepared at 50% w/w %. The feed rate was 9.5 gramsof glucose per liter per hour based on the initial starting volume. At24 hours EFT, IPTG was added to the culture to a concentration of 1 mM.At 49 hours EFT, the fermentation was harvested. The biomass reached66.7 g dry cell weight (DCW)/L at harvest with a fermentation titer of10.4 g IL-29/L fermentor broth.

Example 11 Glucose Fed Batch Fermentation+Yeast Extract Feed ECD920(IL-29 C172S d2-7)

A shake flask (baffled 500 ml flask with 100 ml medium) was preparedwith ZSM medium. Growth was started by inoculating the shake flask witha 0.10 mL of E. coli, ZGOLD1 containing the expression vector pTAP440(EE833) from a vial of a research working cell bank. This vector codesfor a form of the IL-29 molecule that contains a deletion of the secondthrough seventh amino acids. Growth in the shake flask was at atemperature of 32° C. with the agitation set at 250 rpm. The culture wasgrown overnight (16 hours) until the OD₆₀₀ was between 8 and 20 units.

A 6 L fermentation vessel was prepared and sterilized with 3.0 L ofPCOL-18 medium (PCOL-13 medium containing 10.0 g/L of soy peptone).After sterilization the medium was supplemented with glucose at 20 g/L,1 M MgSO₄ (10 ml/L) and kanamycin at 25 ug/ml. The pH of the medium wasadjusted to a pH of 6.8. The vessel aeration was set to 1 vvm andagitation at 350 rpm, temperature was set to 37° C. The fermentor wasinoculated from a shake flask culture that has been grown for 16 hoursand had an OD of 9.6 at 600 nm. The inoculation level was 1%volume/volume. The dissolved oxygen level was maintained above 30%saturation by increasing agitation speed.

A glucose solution (50% w/w) was fed into the fermentor starting at 8hours EFT. The feed rate was 9.5 grams of glucose per liter per hourbased on the initial starting volume. The feed rate was decreased at 24hours EFT to 4.75 g glucose/L/hr (starting volume) and a feed of 25% w/wyeast extract solution was also fed into the fermentor at 4.75 g yeastextract/L/hr (starting volume). At 24 hours EFT, IPTG was added to theculture to a concentration of 1 mM. At 48 hours EFT, the fermentationwas harvested. The biomass reached 74.8 g dry cell weight (DCW)/L atharvest with a fermentation titer of 10.1 g IL-29/L fermentor broth.

Example 12 Glucose Fed Batch Fermentation+Yeast Extract Feed ECD 964(IL-29: d2-7)

A shake flask (baffled 500 ml flask with 100 ml medium) was preparedwith ZSM medium. Growth was started by inoculating the shake flask witha 0.10 mL of E. coli, ZGOLD5 containing the expression vector pTAP440(EE867) from a vial of a research working cell bank. This vector codesfor a form of the IL-29 molecule that contains a deletion of the secondthrough seventh amino acids. Growth in the shake flask was at atemperature of 32° C. with the agitation set at 250 rpm. The culture wasgrown overnight (16 hours) until the OD₆₀₀ was between Band 20 units.

A 6 L fermentation vessel was prepared and sterilized with 3.0 L ofPCOL-18 medium (PCOL-13 medium containing 10.0 g/L of soy peptone).After sterilization the medium was supplemented with glucose at 20 g/L,1 M MgSO₄ (10 ml/L) and kanamycin at 25 ug/ml. The pH of the medium wasadjusted to a pH of 6.8. The vessel aeration was set to 1 vvm andagitation at 350 rpm, temperature was set to 37° C. The fermentor wasinoculated from a shake flask culture that has been grown for 16 hours.The inoculation level was 1% volume/volume. The dissolved oxygen levelwas maintained above 30% saturation by increasing agitation speed.

A glucose solution (50% w/w) was fed into the fermentor starting at 8hours EFT. The feed rate was 9.5 grams of glucose per liter per hourbased on the initial starting volume. The feed rate was decreased at 24hours EFT to 4.75 g glucose/L/hr (starting volume) and a feed of 25% w/wyeast extract solution was also fed into the fermentor at 4.75 g yeastextract/L/hr (starting volume). At 24 hours EFT, IPTG was added to theculture to a concentration of 1 mM. At 48 hours EFT, the fermentationwas harvested. The biomass reached 71.1 g dry cell weight (DCW)/L atharvest with a fermentation titer of 9.8 g IL-29/L fermentor broth.

Example 13 Glucose Fed Batch Fermentation+Yeast Extract Feed ECD 1065(IL-29 C172S d2-7)

A shake flask (baffled 500 ml flask with 100 ml medium) was preparedwith ZSM medium. Growth was started by inoculating the shake flask witha 0.10 mL of E. coli, ZGOLD5 containing the expression vector pTAP440(EE867) from a vial of a research working cell bank. This vector codesfor a form of the IL-29 molecule that contains a deletion of the secondthrough seventh amino acids. Growth in the shake flask was at atemperature of 32° C. with the agitation set at 250 rpm. The culture wasgrown overnight (16 hours) until the OD₆₀₀ was between Band 20 units.

A 6 L fermentation vessel was prepared and sterilized with 3.0 L ofPCOL-18A medium (PCOL 18 medium containing only 10.0 g/L of glucose).After sterilization the medium was supplemented with glucose at 20 g/L,1 M MgSO₄ (10 ml/L) and kanamycin at 25 ug/ml. The pH of the medium wasadjusted to a pH of 6.8. The vessel aeration was set to 1 vvm andagitation at 350 rpm, temperature was set to 37° C. The fermentor wasinoculated from a shake flask culture that has been grown for 16 hoursand had an OD of 11.8 at 600 nm. The inoculation level was 1%volume/volume. The dissolved oxygen level was maintained above 30%saturation by increasing agitation speed.

A glucose solution (50% w/w) was fed into the fermentor starting at 8hours EFT. The feed rate was 4.0 grams of glucose per liter per hourbased on the initial starting volume from 8-9 hours EFT. AT 9 hours EFT,the feed rate was increased to 8.0 grams of glucose per liter per hourbased on the initial starting volume. The feed rate was decreased at 24hours EFT to 4.0 g glucose/L/hr (starting volume) and a feed of 25% w/wyeast extract solution was also fed into the fermentor at 4.0 g yeastextract/L/hr (starting volume). At 24 hours EFT, IPTG was added to theculture to a concentration of 1 mM. At 47 hours EFT, the fermentationwas harvested. The biomass reached 66.9 g dry cell weight (DCW)/L atharvest with a fermentation titer of 9.3 g IL-29/L fermentor broth.

Example 14 Direct Homogenization and Batch Centrifugation ofFermentation Broth

Fermentation broth (0.90 L) from ECD892 (described above) was mixed with0.90 L of deionized water. The diluted broth mixture was passed throughan APV-Gaulin 2000 homogenizer at 10,000 psi. The outlet of thehomogenizer was connected with a heat exchanger hooked up to are-circulating water bath set at 2-8° C. The mixture was collected afterpassing through the homogenizer and was passed through a second time at10.00 psi. This process was repeated a third and final time. The IL-29yield in the homogenate was 10.9 g/L fermentor broth for an 87% processyield

The homogenate was transferred to 1.0 L Beckman centrifuge bottles,adding 0.90 L of homogenate per bottle. The mixture was then centrifugedfor 30 minutes in a Beckman Avanti JHC centrifuge at 15,000×g and 4° C.using a Beckman JLA-8.1 fixed angle rotor.

After centrifuging water was added in an equal volume to the supernatantdecanted. The pellet was re-suspend by mixing with a spatula followed bymixing with an Omni EZ hand held homogenizer. The mixture washomogenized using the appropriate sized metal probe at half power for˜15 seconds or until the cell pellet was well suspended. The mixture wasthen re-centrifuged at 15,000×G using Beckman Avanti JHC centrifuge withthe Beckman JLA-8.1 fixed angle rotor for 30 minutes at 4° C. Thisprocess was repeated an additional time and the resulting washedinclusion body (WIB) pellet was ready for refolding. The WIB yield wasequalized to 9.6 g IL-29 per liter fermentor broth. The step yield fromhomogenate was 88% and the overall yield from fermentor broth was 76.8%.

Example 15 Direct Homogenization and Continuous Centrifugation ofFermentation Broth

Fermentation broth (80.0 Kg) from ECD967 (Il-29=8.0 g/L) was mixed with80.0 Kg of deionized water. The diluted broth mixture was passed througha Niro-Sovai homogenizer at 800 bar. The outlet of the homogenizer wasconnected with a heat exchanger hooked up to chilled water set at 2-8°C. The mixture was collected after passing through the homogenizer andwas passed through a second time at the same pressure. This process wasrepeated a third and final time. The IL-29 yield in the homogenate was7.24 g/L fermentor broth for a 90% process yield.

The homogenate containing 3% solids (wt/wt) was then centrifuged andwashed on a Westfalia C6 disc stack centrifuge. The solution was passedinto the centrifuge at 1.5 L/minute with the G force set at 15,000×g.The solids were kept in the bowl, while the supernatant stream wascontinuously discharged. At a predetermined set point, the homogenatefeed was stopped and purified water was passed over the solids at 1.5L/minute. This water wash displaced supernatant in the bowl. The solidsand water in the bowl of the centrifuge were then discharged. Thedischarged solids (19.81 Kg) contained 17.6% solids (wt/wt) while thesupernatant contained approximately 1% solids. The discharged solidswere split into 8 containers each containing 2.4 Kg of material.

Example 16 Solubilization of WIB Pellets Using Guanidine Solutions

A WIB pellet was produced from fermentation ECD917 as described inExample 14. The wet weight of the WIB pellet from 2 liters offermentation broth was approx. 400 g. A 6M Guanidine HCl solutioncontaining 40 mM dithiothreitol was prepared as described in Table 4below. Three hundred milliliters of this solution was added to the WIBpellet and the pellet was distributed into the solution using a smallhand held homogenizer. The solution was allowed to solubilized for 1hours at room temperature. After solubilization, 700 ml of solute wasobtained. This material had an IL-29 content of 19.5 mg/ml and thesolute contained 13.65 g of IL-29. This was equivalent to 6.82 g ofIL-29 per liter fermentation broth and the process yield was 70%.

TABLE 4 Guanidine HCl/DTT solution Formula Component Weight Amount/L 2 MTris Stock 50 mL pH 8.0 Guanidine HCl  95.53 g/mol 573.18 g DTT 154.25g/mol 3.09 g Deionized Water QS to 1000 mL

Example 17 Solubilization of Discharged Solids Using Guanidine Powders

Tris base (11.0 g) and Tris HCl (23.4 g) powders were added to 2.4 Kg ofECD967 discharged solids as described in Example 15. The powders weremixed into solution and the pH adjusted to 8.0. Dithiothreitol (14.8 g)and guanidine HCl (1.37 Kg) were added to the mixture. The solution wasallowed to mix for 1 hour without temperature adjustment. Aftersolubilization the mixture weighed 3.78 Kg and contained 55.72 gramsIL-29.

Example 18 Refolding of Solubilized WIB Pellets Using Cysteine andCystine

A 5.0 L glass refold vessel was filled with 3.0 L of 1.1 M Arginine HClbuffer and 0.167 L of 20× salts (see Table 5 below). A stir bar wasadded to the vessel and the vessel was placed on a stir plate. This unitwas then placed into a refrigerated incubator at 8° C. with mixing setat a low speed. To this solution 0.77 g DTT and 0.167 L of 120 mMcystine solution (See Table 6 below) were added. This mixture is used tomake a cysteine and cystine redox pair at a ratio of 6:1. The pH wasadjusted to 8.0 with NaOH. After preparation, 0.3 L of buffer wasremoved from the vessel and replaced with 300 ml of the solute solutionfrom ECD917 as described above in Example 16. The 300 ml of solutecontained 5.85 g of IL-29 and the starting concentration of unfoldedIL-29 was 1.95 mg/mL. The solution was allowed to refold for 6 hours andwas stopped by adjusting the pH to 5.5 with 20% acetic acid. The finalconcentration of refolded was 1.12 mg/mL. The refolding yield was 57%and produced 3.4 g of refolded IL-29.

TABLE 5 1.1 M Arginine buffer solution Formula Amount Component WeightMolarity [g or mL/L] 2 M Tris stock N/A 0.05 M 27.5 mL pH 8.0 ArginineHCl 210.67  1.1 M 231.7 PEG 3400 3350 0.55 g Deionized Water QS to 1000mL

TABLE 6 120 mM Cystine solution prepared in 0.25 M NaOH solution FormulaAmount Component Weight [g or mL/L] L-Cystine 240.3 g 28.8 g 10 M NaOHsolution N/A 25.0 mL to make 0.25M NaOH Water QS to 1000 mL

Example 19 Refolding of Discharged Solids Using Cysteine and Cystine

Refold buffer containing 1.1 M arginine HCl was prepared as describedabove in Table 5. Salts solutions (20×) and 120 mM cystine solution werealso prepared as described below in Table 7. Arginine buffer (30.0 L)was dispensed into a 50 L jacketed tank with agitation (100 rpm) andcooling set at 8° C. To the arginine buffer solution, 1.67 L of 20 saltsand 1.67 L of 120 mM cystine solution were added. Dithiothreitol (7.7 g)was added and the pH was adjusted to pH 8.0 with 10 N NaOH. The solute(3.35 L) prepared in Example 17 was added to the mixture over a 4 hourperiod. The starting refold concentration of unfolded IL-29 was 1.72mg/ml. The refold was allowed to proceed for an additional 2 hoursbefore stopping the reaction by adding 20% acetic acid until the pH waslowered to 5.5. The solution was diluted by adding 120 L of 25 mMacetate buffer pH 5.5. The solution was allowed to settle overnight atroom temperature. The mixture was filtered using a Cuno BioPlus filter.The IL-29 concentration after refolding was 0.90 mg/mL. The IL-29concentration in the diluted and clarified solution was 0.19 mg/mL. Theoverall refolded IL-29 in the clarified broth was 29.85 g for a 54%refolding yield.

TABLE 7 20 X salts solution Component Molarity of Solution 20 X saltstock solution 0.20 M NaCl 0.04 M MgCl₂—6H₂O 0.01 M KCl 0.04 MCaCl₂—2H₂O

Example 20 Clarification and Capture of Renatured IL-29

Reducing the pH to 6.0 quenches the IL-29 refolding reaction. Theacidified refold is diluted 4.25-fold in 25 mM sodium acetate, pH 5.6 toprecipitate misfolded and unfolded proteins and to condition the refoldfor loading to the capture column. The precipitate is allowed to settleovernight and then the supernatant is clarified through a depth filtertrain composed of a coarse Cuno Zeta Plus Maximizer 30M03 (nominal 0.8μm) and fine Cuno Zeta Plus Maximizer 90M05 (nominal 0.2 μm) filter inseries.

The clarified, diluted IL-29 is captured on a ToyoPearl SP550C (TosohBioscience) cation exchange column at pH 5.5. In this case, a 14.5 cmtall×14 cm diameter column (2.23 L column volume) is used at 180 cm/hrto capture renatured IL-29 C172S d2-7 originating from 10 L offermentation broth. The column is equilibrated with 50 mM sodiumacetate, pH 5.5, and then loaded with 29 g IL-29 (13 g/L resin). Afterwashing with equilibration buffer, bound IL-29 is eluted with anincreasing linear gradient to 2 M sodium chloride, in 50 mM sodiumacetate, pH 5.5, over 5 column volumes. Based on absorbance at 280 nm,one pool of eluted material is collected from 0.2 AU to 1.0 AU on thefront side of the elution peak. A second pool is collected from 1.0 AUon the front side to 0.2 AU on the trailing edge of the elution peak.The first pool, comprised mostly of non-IL-29 proteins, is discarded.The second pool is carried forward for intermediate purification.Similar methods have been used to capture other IL-29 variants,including the native, the C172S, and the C172S Leu insert forms.

Alternatively, an isocratic salt elution may be used to displace IL-29from the column. In this example, a 10 cm tall×1.6 cm diameter column ofToyoPearl SP550C resin is used at 180 cm/hr to capture renatured C172SIL-29 originating from 4 L of fermentation broth. The column isequilibrated with 50 mM sodium acetate, pH 5.5, and then loaded with 136mg IL-29 (6.8 g/L resin). After washing with equilibration buffer, boundIL-29 is eluted with a 10 CV step at 600 mM NaCl, in 50 mM sodiumacetate, pH 5.5. Based on absorbance at 280 nm, material eluted from˜20% of maximal signal on the upslope of the peak to ˜20% of the maximalsignal on the downslope is pooled and carried forward for intermediatepurification. Substantially similar step elution methods have also beenused to capture and elute the IL-29 C172 d2-7 variant.

Example 21 Hydrophobic Interaction Chromatography Using Super Butyl 550Cresin

A capture pool containing the IL-29 C172S d2-7 protein was adjusted to1.0 M (NH₄)₂SO₄ by performing a 2-fold dilution with 50 mM sodiumacetate, 2.0 M (NH₄)₂SO₄, pH 5.5. This solution was passed through a0.45 μm filter to remove insoluble material. The filtered andconditioned HIC load solution was applied to a ToyoPearl Super Butyl550C (Tosoh Bioscience) column, previously equilibrated with 50 mMsodium acetate, 1.5 M (NH₄)₂SO₄, pH 5.5. Here, a 14 cm tall×14 cmdiameter column (2.16 L CV) was operated at 150 cm/hr and at roomtemperature to purify IL-29 originating from 10 L of fermentation broth(12.4 g IL-29 loaded per liter resin). The HIC column was washed withequilibration buffer to remove unbound material and then IL-29 waseluted with a linear gradient to 50 mM sodium acetate, pH 5.5, over 10column volumes (CV). Based on absorbance at 280 nm, ⅓ CV fractions werecollected from 0.1 AU on the leading edge to 0.1 AU on the trailing edgeof the elution peak. Measurements of 280 nm absorbance were collectedfor each fraction, and the fraction with maximal A280 identified. Forpooling, fractions containing at least 20% of the maximal OD280 on theup slope to those containing at least 45% of the maximal OD280 on thedown slope are combined.

Example 22 Hydrophobic Interaction Chromatography Using Butyl 650M Resin

A capture pool containing the IL-29 C172S d2-7 protein was adjusted to1.0 M (NH₄)₂SO₄ by performing a 2-fold dilution with 50 mM sodiumacetate, 2.0 M (NH₄)₂SO₄, pH 5.5. This solution was passed through a0.45 μm filter to remove insoluble material. The filtered andconditioned HIC load solution was applied to a to a ToyoPearl Butyl 650M(Tosoh Bioscience) column, previously equilibrated with 50 mM sodiumacetate, 1.5 M (NH₄)₂SO₄, pH 5.5. Here, a 11 cm tall×10 cm diametercolumn (0.86 L CV) was operated at 150 cm/hr and at room temperature topurify IL-29 originating from 4 L of fermentation broth (8.6 g IL-29loaded per liter resin). The HIC column was washed with equilibrationbuffer to remove unbound material and then IL-29 was eluted with alinear gradient to 50 mM sodium acetate, pH 5.5, over 10 column volumes(CV). Substantially similar methods have been used to purify the C172SLeu Insert variants of IL-29 on Butyl 650M resin.

Example 23 Hydrophobic Interaction Chromatography Using Phenyl 650MResin

A capture pool containing the IL-29 C172S protein was adjusted to 1.0 M(NH₄)₂SO₄ by performing a 2-fold dilution with 50 mM sodium acetate, 2.0M (NH₄)₂SO₄, pH 5.5. This solution was passed through a 0.45 μm filterto remove insoluble material. The filtered and conditioned HIC loadsolution was applied to a ToyoPearl Phenyl 650M (Tosoh Bioscience)column, previously equilibrated with 50 mM sodium acetate, 1.5 M(NH₄)₂SO₄, pH 5.5. Here, a 10 cm tall×10 cm diameter column (0.785 L CV)was operated at 150 cm/hr and at room temperature to purify IL-29originating from 5 L of fermentation broth (7.0 g IL-29 loaded per literresin). The HIC column was washed with equilibration buffer to removeunbound material and then IL-29 was eluted with a linear gradient to 50mM sodium acetate, pH 5.5, over 10 column volumes (CV). Substantiallysimilar methods have been used to purify the native IL-29 protein onPhenyl 650M resin.

Example 24 Hydrophobic Interaction Chromatography Using Other HIC Resins

A capture pool containing the native IL-29 protein was adjusted to 1.5 M(NH₄)₂SO₄ by performing a 2-fold dilution with 50 mM sodium acetate, 3.0M (NH₄)₂SO₄, pH 5.5. This HIC load solution was passed through a 0.45 μmfilter to remove insoluble material. The filtered and conditionedsolution was divided and loaded to six separate columns, each previouslyequilibrated with 50 mM sodium acetate, 1.5 M (NH₄)₂SO₄, pH 5.5. Resinstested include: Ether 650M (Tosoh Bioscience), PPG 600M (TosohBioscience), Octyl Sepharose (GE Healthcare), Phenyl Sepharose 6 FastFlow (low substitution version, GE Healthcare), Butyl Sepharose 4 FastFlow (GE Healthcare), and Phenyl Sepharose 6 Fast Flow (highsubstitution version, GE Healthcare). Here, each 8 cm tall×1.6 cmdiameter column (16 mL CV) was operated at 150 cm/hr and at roomtemperature to purify IL-29 at a 5 g IL-29 per liter resin load factor.Each HIC column was washed with equilibration buffer to remove unboundmaterial and then IL-29 was eluted with a linear gradient to 50 mMsodium acetate, pH 5.5, over 10 column volumes (CV).

Example 25 Purification of IL-29 by High Performance Cation ExchangeChromatography

A HIC eluate pool containing the IL-29 C172S d2-7 was diluted 6-fold inwater then 0.2 μm filtered and applied to an SP Sepharose HP (GEHealthcare) column, equilibrated in 50 mM sodium acetate, 300 mM sodiumchloride, pH 5.5. Here, a 16 cm tall×10 cm diameter column (1.26 L CV)was operated at 125 cm/hr to purify IL-29 after loading to a 15.6 gIL-29 per liter resin load factor. The high performance cation exchangecolumn was washed with equilibration buffer and then eluted with a 20 CVlinear gradient to 50 mM sodium acetate, 800 mM sodium chloride, pH 5.5.Based on absorbance at 280 nm, one-third CV fractions were collectedfrom 0.1 AU on the leading edge to 0.1 AU on the trailing edge of theelution peak. Measurements of 280 nm absorbance were collected for eachfraction, and the fraction with maximal A280 identified. For pooling,fractions containing at least 80% of the maximal OD280 on the up slopeto those containing at least 20% of the maximal OD280 on the down slopewere combined. Similar methods have been used to purify the C172S andthe C172S Leucine Insert forms of IL-29.

Alternatively, an isocratic salt elution may be used to displace IL-29from the column. In this example, a 15.8 cm tall×1.6 cm diameter column(31.6 mL CV) of SP Sepharose HP (GE Healthcare) is used at 150 cm/hr topurify IL-29 from diluted and filtered HIC Pool. The column isequilibrated with 50 mM sodium acetate, 300 mM sodium chloride, pH 5.5,and then loaded with 304 mg IL-29 (9.6 g IL-29 per L resin). Afterwashing with equilibration buffer, bound IL-29 is eluted with a 10 CVstep at 450 mM NaCl, in 50 mM sodium acetate, pH 5.5.

Example 26 Purification of IL-29 Using Other Cation Exchange Resins

A capture pool containing native IL-29 was diluted in 50 mM sodiumacetate, pH 5.5 to a conductivity of <40 mS/cm, then filtered togenerate the column load. The filtered and conditioned solution wasdivided and loaded to two separate columns, each previously equilibratedwith 50 mM sodium acetate containing sodium chloride, at pH 5.5. Resinstested include: CM Sepharose Fast Flow (GE Healthcare), and FractogelSO₃ ⁻ (EMD Biosciences). Here, each 8 cm tall×1.6 cm diameter column(16.1 mL CV) was operated at 150 cm/hr and at room temperature to purifyIL-29 after column loading at a 5 g IL-29 per liter resin load factor.Each cation exchange column was washed with equilibration buffer toremove unbound material and then IL-29 was eluted with a linear gradientof increasing salt concentration in 50 mM sodium acetate, pH 5.5, over20 column volumes (CV). For the CM Sepharose column the gradient spannedfrom 100 to 800 mM sodium chloride, while for the Fractogel resin thegradient was from 400 mM to 2 M sodium chloride.

Example 27 Purification of IL-29 by Hydrophobic Charge InductionChromatography

A HIC eluate pool containing IL-29 C172S was concentrated and bufferexchanged into 50 mM Tris, 100 mM NaCl, pH 8. The material was appliedto a mercaptoethyl pyridine (MEP) HyperCel (BioSepra) column, previouslyequilibrated with 50 mM Tris, 100 mM NaCl, pH 8. Here, a 6 cm tall×1.1cm diameter column (5.7 mL CV) was operated at 90 cm/hr to purify IL-29at an ˜10 g IL-29 per liter resin load factor. The MEP HyperCel resinwas washed with equilibration buffer at pH 8 then washed with acitrate-phosphate buffer at pH 6.5. Recombinant IL-29 was then elutedfrom the HCIC resin with a 10 CV linear gradient to citrate-phosphatebuffer at pH 4.5.

Example 28 Purification of IL-29 by Immobilized Metal AffinityChromatography

A capture pool containing the native form of IL-29 was applied to aChelating Sepharose (GE Healthcare) column previously charged withcopper (from cupric sulfate) and equilibrated in 50 mM sodium acetate,800 mM sodium chloride, pH 5.5. The 8 cm tall×1.6 cm diameter column(16.1 mL CV) was operated at 150 cm/hr to purify IL-29 at an ˜5 g IL-29per liter resin load factor. The copper chelated resin was washed withequilibration buffer, and IL-29 eluted with a 10 CV linear gradient to abuffer containing 25 mM sodium acetate, 800 mM sodium chloride, 500 mMimidazole, pH 5.5. Similar results were obtained when either nickel(from nickel sulfate) or zinc (from zinc chloride) was used as thechelated divalent cation.

Example 29 Concentration of Purified IL-29 Bulk Intermediate

The SPHP pool was concentrated approximately 2-3 fold using a 5 kDamolecular weight cut-off polyether sulfone tangential flow filtration(TFF) plate and frame membrane at a transmembrane pressure of ˜20 psi.For the 10 L scale process described here, a membrane surface area of0.1 m² and an inlet flow rate of 15 L/hr was used. After the retentatehas been concentrated to ˜15 mg/mL, it was removed from the TFF system,the system was rinsed with 50 mM sodium acetate, pH 5.5. The rinse wascombined with the concentrated retentate to achieve a final ˜12.5 mg/mLconcentration. This solution is then filtered through a 0.2 μm membrane,then filled in appropriate containers and stored at ≦−60° C. inpreparation for the subsequent PEGylation reaction.

Example 30 PEGylation of IL-29 with 20 kDa Linear mPEG-Propionaldehyde

In preparation for a PEGylation reaction, IL-29 bulk intermediate wasthawed and transferred to a reaction vessel. Buffer for dilution, 100 mMsodium cyanoborohydride reductant stock solution, and 100 g/Lderivatized PEG (20 kDa linear methoxyPEG-propionaldehyde) stocksolution, was added to the reaction to create a mixture with 5 g/LIL-29, 10 g/L derivatized PEG (2 PEG per IL-29 on a molar basis), and 20mM sodium cyanoborohydride at pH 5.5 in 50 mM sodium acetate buffer. Inthe current example, 16 g of IL-29 bulk intermediate at 13.54 g/L (1.18L volume) was mixed with 1.06 L of 50 mM sodium acetate, pH 5.5, 0.64 Lof 100 mM reductant stock, and 0.32 L of 100 g/L PEG stock to make a 3.2L volume with the reaction parameters described above. The reaction wasallowed to proceed with mixing for ˜18 hr at 20° C. under subduedlighting. These reaction conditions resulted in a mixture of 65-75%monoPEGylated IL-29, with 10-20% each of the unPEGylated andmulti-PEGylated species, when using the C172S Leucine Insert or C172Sd2-7 form of recombinant IL-29 as starting material. Similar resultswere also obtained when IL-29 at a 3 g/L concentration was reacted with6 g/L derivatized PEG (2 PEG per IL-29 on a molar basis), and 20 mMsodium cyanoborohydride at pH 5.5 in 50 mM sodium acetate buffer.

Example 31 PEGylation of IL-29 with 30 kDa Linear mPEG-Propionaldehyde

In preparation for a PEGylation reaction, IL-29 bulk intermediate wasthawed and transferred to a reaction vessel. Buffer for dilution, 100 mMsodium cyanoborohydride reductant stock solution, and 150 g/Lderivatized PEG (30 kDa linear methoxyPEG-propionaldehyde) stocksolution, were added to the reaction to create a mixture with 5 g/LIL-29, 15 g/L derivatized PEG (2 PEG per IL-29 on a molar basis), and 20mM sodium cyanoborohydride at pH 5.5 in 50 mM sodium acetate buffer. Inthis example, 2.5 mg of IL-29 C172S bulk intermediate at 12.8 g/L (195μL, volume) was mixed with 155 μL, of 50 mM sodium acetate, pH 5.5, 100μL, of 100 mM reductant stock, and 50 μL, of 150 g/L PEG stock to make a0.5 mL volume with the reaction parameters described above. The reactionwas allowed to proceed with mixing for ˜18 hr at 20° C. under subduedlighting. These reaction conditions resulted in a mixture withcomparable levels of monoPEGylated IL-29 vs. a 5 g/L IL-29, 2:1PEG:protein reaction with the 20 kDa version of mPEG-propionaldehyde.

Example 32 PEG-IL-29 Purification by High Performance Cation ExchangeChromatography

After the PEG reaction was completed, the reaction mixture was diluted2-fold with 50 mM sodium acetate, pH 5.5 then 0.2 μm filtered and loadedto a SP Sepharose HP (GE Healthcare) column, equilibrated in 50 mMsodium acetate, 200 mM sodium chloride, pH 5.5. In this example, a 16 cmtall×10 cm diameter column (1.26 L CV) was operated at 125 cm/hr topurify PEG-IL-29 originating from a PEG reaction using 8 g of IL-29. Thehigh performance cation exchange column was washed with equilibrationbuffer and then eluted with a 10 CV linear gradient to 50 mM sodiumacetate, 500 mM sodium chloride, pH 5.5. Based on absorbance at 280 nm,one-third CV fractions were collected from 0.1 AU on the leading edge to0.1 AU on the trailing edge of the elution peak. Fractions were analyzedfor monoPEG-IL-29 content by reversed phase HPLC, and those fractionscontaining at least 99% mono-PEGylated IL-29 were pooled. Similarresults were obtained regardless of whether the PEG-IL-29 was derivedfrom the C172S Leucine Insert or C172S d2-7 form of the molecule.

PEG-IL-29 may also be eluted from the SP HP column using isocraticmethods. In this example, a reaction mixture using IL-29 C172S d2-7 wasdiluted 2-fold with 50 mM sodium acetate, pH 5.5 then 0.2 μm filteredand loaded to a SP Sepharose HP (GE Healthcare) column, equilibrated in50 mM sodium acetate, 200 mM sodium chloride, pH 5.5. Here, a 15.5 cmtall×1.6 cm diameter column (31 mL CV) was operated at 125 cm/hr topurify PEG-IL-29 after loading the column at a 9 g IL-29 per liter resinload factor. The high performance cation exchange column was washed with5 CV equilibration buffer and with a 3 CV step at 50 mM sodium acetate,240 mM sodium chloride, pH 5.5. PEG-IL-29 was then eluted with a 4 CVstep at 400 mM sodium chloride, in 50 mM sodium acetate, pH 5.5.

Example 33 Ultrafiltration/Diafiltration of PEG-IL-29

The SP HP eluate pool containing monoPEGylated IL-29 was concentrated byultrafiltration in a tangential flow filtration system equipped with a 5kDa molecular weight cut-off polyether sulfone plate and frame membraneat a trans-membrane pressure of ˜20 psi. For a 7.7 g scale processdescribed here, a membrane surface area of 0.1 m² and an inlet flow rateof 15 L/hr was used. After the retentate has been concentrated to ˜15-20mg/mL, it was diafiltered against 7 diavolumes of formulation buffer.The formulated bulk was removed from the TFF system, and the system wasrinsed with formulation buffer. The rinse was combined with theconcentrated retentate to achieve a final ˜12-14 mg/mL concentration.This solution was then filtered through a 0.2 μm membrane, then filledin appropriate containers and stored at ≦−60° C. to generate PEG-IL-29bulk drug substance.

The complete disclosure of all patents, patent applications, andpublications, and electronically available material (e.g., GenBank aminoacid and nucleotide sequence submissions) cited herein are incorporatedby reference in their entirety. The foregoing detailed description andexamples have been given for clarity of understanding only. Nounnecessary limitations are to be understood therefrom. The invention isnot limited to the exact details shown and described, for variationsobvious to one skilled in the art will be included within the inventiondefined by the claims.

1. A method of recovering an IL-29 polypeptide comprising: (a) providingan ompT deficient and fhuA deficient E. coli host cell comprising anexpression vector comprising the following operably linked elements: (i)a transcription promoter; (ii) a nucleic acid molecule encoding an IL-29polypeptide comprising amino acid residues 1-176 of SEQ ID NO:6; and(iii) a transcription terminator; in a suitable growth medium; (b)adding an inducing agent to induce expression of the IL-29 polypeptide;(c) harvesting the host cells; (d) lysing the host cells; (e)centrifuging the lysed host cells; (f) recovering the inclusion bodypellet; (g) solubilizing the inclusion body pellet; (h) adding thesolubilized IL-29 polypeptide to a refolding buffer pH7.3-8.5 to a finalconcentration of 0.5-3.0 mg/ml, said refolding buffer comprises0.05%-0.5% polyethylene glycol, 0.5-1.25 M arginine, and a mixture ofreduced and oxidized molecule, wherein the solubilized IL-29 polypeptideis refolded for 1-26 hours at a temperature of 4-30° C.; (i) quenchingthe refolding reaction by adjusting the pH to 5.5-6.5; (j) diluting thequenched refolding solution 1.5- to 10-fold in water or low ionicstrength buffer at pH 5-7; and (k) filtering the quenched, dilutedrefolding solution through filters to remove precipitate orparticulates.
 2. The method of claim 1, wherein the expression vector ofstep (a) further comprises a translational enhancer.
 3. The method ofclaim 2, wherein the translational enhancer is the nucleic acid sequenceof SEQ ID NO:13.
 4. The method of claim 1, wherein the inducing agent ofstep (b) is isopropyl thiogalactopyranoside.
 5. The method of claim 4,wherein the isopropyl thiogalactopyranoside is added to the growthmedium at a concentration of 0.5 mM to 2 mM.
 6. The method of claim 1,wherein the host cells of step (c) are harvested by centrifugation. 7.The method of claim 1, wherein the host cells of step (d) are lysed byhomogenization.
 8. The method of claim 1, wherein the lysed host cellsof step (e) are centrifuged by either batch or continuouscentrifugation.
 9. The method of claim 1, wherein the inclusion bodypellet of step (g) is solubilized in 4-6 M guanidine hydrochloride and10-50 mM dithiothreitol for 1-2 hours at 15-25° C.
 10. The method ofclaim 1, wherein the mixture of reduced and oxidized molecules of therefolding buffer are selected from the group consisting of cysteine andcystine, dithiothreitol and cystine, reduced glutathione and oxidizedglutathione, and dithiothreitol and oxidized glutathione.
 11. The methodof claim 1, wherein the filter of step (k) is a depth filter.